key: cord- -leedutqo authors: nawaz, sameena; allen, david j.; aladin, farah; gallimore, christopher; iturriza-gómara, miren title: human bocaviruses are not significantly associated with gastroenteritis: results of retesting archive dna from a case control study in the uk date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: leedutqo gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. despite improvements in detection methods, a significant diagnostic gap still remains. human bocavirus (hbov)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between hbovs, and in particular type- hbovs, and gastroenteritis has previously been made. the aim of this study was to determine the role of hbovs in gastroenteritis, using archived dna samples from the case-control infectious intestinal disease study (iid). dna extracted from stool samples from , cases and , controls were tested for the presence of hbov dna. all samples were screened in a real time pcr pan-hbov assay, and positive samples were then tested in genotype to -specific assays. hbov was detected in . % but no significantly different prevalence was observed between cases and controls. in the genotype-specific assays of the hbov-positive samples were genotyped, with hbov- predominantly found in controls whilst hbov- was more frequently associated with cases of gastroenteritis (p< . ). a significant proportion of hbov positives could not be typed using the type specific assays, % of the total positives, and this was most likely due to low viral loads being present in the samples. however, the distribution of the untyped hbov strains was no different between cases and controls. in conclusion, hbovs, including hbov- do not appear to be a significant cause of gastroenteritis in the uk population. in a novel parvovirus was discovered in respiratory secretions of young children and was termed human bocavirus (hbov- ) [ ] . other important members of the parvoviridae family include b which causes fith disease and human parvovirus (parv ) which has not yet been associated with a disease [ ] . parvoviruses in animals are generally associated with systemic disease but also with respiratory and enteric symptoms [ , ] . since the discovery of hbov- three other hbov genotypes have been described, hbov- , hbov- and hbov- . the association between hbov- and respiratory disease has previously been well established [ , , , , , , , , , , , , , , ] . although all hbovs have also been detected in stool samples with prevalences ranging from , % to %, only hbov- has been reported to be associated with symptoms of gastroenteritis [ ] . nevertheless, the role of hbov as an aetiological agent of gastroenteritis has not been clearly confirmed, furthermore, to date, no clear association between the presence of hbov- and hbov- and disease has been established [ , ] . recent seroepidemiological studies indicate that exposure to hbovs occurs early in life and % of the population are seropositive by the age of , although differences were reported in the seroprevalence of type-specific antibodies to the different hbovs, which suggested that hbov- infections are more prevalent [ ] . the infectious intestinal disease study (iid study) was a large case control study of gastroenteritis carried out in the uk between - [ ] with the aim to determine the burden and aetiology of sporadic cases iid in the uk population. initially, the use of classical microbiology diagnostic methods and electron microscopy (for virus detection) failed to detect a potential aetiological agent or toxin in % of the cases [ ] . retesting of the archived samples from this study using molecular methods for the detection of enteric viruses, bacteria and protozoa revealed viruses to be the most common aetiological agents of gastroenteritis,the diagnostic gap for iid was reduced to % from % [ ] . the aim of the present study was to evaluate the role of hbovs in iid in the uk population, using archived dna samples from the matched case-control iid- study [ ] . in addition, the presence specifically of hbov- , or was investigated in order to determine any possible associations between specific hbov genotypes and iid. a total of , archived dna from the iid study [ , ] were tested for the presence of hbov dna. this archive comprised dna extracted from stool samples from , cases and , controls. the qpcr assay targeted the ns gene (ratcliff et al., unpublished method, personal communication) and was performed using an abi taqman . oligonucleotide primer and probe sequences and positions are described in table . the reaction consisted of . m ddt (invitrogen), x platinum quantitative pcr supermix-udg (invitrogen), pan-hbov-f and pan-hbov-r primers each at a concentration of mm, pan-hbov-ns probe at mm concentration, rox mm (invitrogen) . ml of template dna and rnase free water to a final reaction volume of ml. the amplification consisted of an initial denaturation at uc for min, followed by cycles with denaturation at uc for sec, annealing at uc for sec and extension at uc for sec. hbov- , and -specific primer pair and probes were designed in house through alignment of sequence data available in genbank. the reaction conditions are as follows; x platinum quantitative pcr supermix-udg (invitrogen), hbov-ns - f, hbov- r, hbov -r and hbov r primers each at a concentration of mm, the hbov , and probe at mm concentration, rox mm (invitrogen), . ml of template and rnase free water was added to a final reaction volume of ml. the amplification conditions for the typing assay are the same as those described in the hbov ns detection assay above. plasmids containing a bp and a bp region of the ns encoding gene of hbov- and hbov- respectively were used for assay optimisation and as controls. control material was kindly provided by r. ratcliff, adelaide, australia. the controls were also used in order to generate a standard curve for use with the pan-hbov assay in order to allow for normalisation of the data generated including the comparison of relative sensitivities of the different assays and for quantitation of dna present in each of the positive samples. the standard curve was generated using the plasmid containing a genome segment of the hbov- and consisted of a series of fold dilution containing from , copies/ml down to copies/ml. inter-and intraassay reproducibility was analysed by performing replicate testing of the standards in a single run (x ) and repeated runs (x ), and the standard curve was also included in each assay run for quality control and normalisation of results. a subset of samples positive in the pan-hbov assay but which failed to amplify in the type-specific assays were confirmed using an alternative method published elsewhere [ , ] , and were further confirmed though direct sequencing of the amplicons obtained after purification either from solution or agarose gels using agencourt ampure (beckman coulter, usa) and geneclean spin kit (qbiogene), respectively, following manufactures protocols. the chi-squared test was used in order to evaluate the significance of differences observed between groups. for comparison of median values (analysis of ct values) the mann witney utest was used. prevalence odds ratio (por = pcases/( -pcases)/ pcontrols/( -pcontrols)) was calculated in the total cohorts and by age group. table . hbov-specific oligonucleotide primers and probes (all located at the ns gene). sequence ( a total of . % of the samples tested were positive for hbov. no statistically significant differences were seen in the prevalence of hbov between cases and asymptomatic controls, por = . (table ) . peak hbov infection was observed in children under the age of , both in cases and controls, with significantly higher hbov incidence in children between and in asymptomatic controls than in the cases of gastroenteritis (por = . ; p, . ). the number of hbov positives in older age groups was too small for meaningful statistical analysis. the average ct values were . and . , and the median ct values were . and . in cases and controls respectively (see distribution in figure ). the majority of hbov-positives in both cases and controls had copy numbers ranging between and copies/reaction (or between . and . copies/ml of feaces). the distribution of hbov viral loads between cases and controls was comparable and the median ct values between cases and controls were not significantly different (u-test; z = . , p. . ). hbov dna was found in ( %) samples in the absence of other co-pathogens (table ) . no statistically significant differences were observed in the proportion of cases or controls in which hbov was found as a single organism or in the presence of one or more pathogens in the cohort as a whole, however in the - years of age group, hbov in the absence of any other enteric pathogens was found in % of the cases, but in % of the controls (p, . ). hbov infections were detected year round although a peak was observed in the spring/early summer months, between april and june (figure ). hbov dna was found in . % and . % of female cases and controls, respectively. the distribution of hbov among females and males was not significantly different from the distribution of females and males in the entire cohort which was % and %, respectively. a total of ( . %) hbov positives were genotyped, whilst ( . %) remained untyped after testing in the hbov types , or specific assays (table ). hbov- detection was found predominantly in controls, (p, . ) and hbov- was predominantly associated with cases (p, . ). the prevalence of hbov- was not significantly different between cases and controls. hbov- and - were predominantly found in children (table ) . hbov- in the absence of any other pathogen was detected in ( . %) of the cases, compared to ( . %) of the controls. in cases, hbov- was found across the age groups, although more frequently in children , , whereas in controls they were found predominantly in children , with only example in an adult ( table ). the prevalence of hbov- in children , years old was however not significantly different between cases and controls, . % and . %, respectively. a subset of hbov that were negative in the type , or specific assays were confirmed in an alternative pan-hbov pcr, and sequencing of a small number confirmed them as types , or . the majority of the untyped samples ( %) had a ct value of . in the screening pan-hbov pcr, indicative of low viral loads being present in the samples. this represents the largest study to date investigating the role and distribution of hbovs infections in community acquired sporadic gastroenteritis and in asymptomatic controls. the prevalence of hbov infection in the uk population was found to be . % across all ages, with a higher percentage of the infections occurring in children , years of age ( %). however, the prevalence of hbov infections was comparable in cases of gastroenteritis and in age-matched asymptomatic controls. although the presence of enteric pathogens, eg norovirus or rotavirus, in asymptomatic individuals is well documented, a significantly higher prevalence of the pathogen is seen in cases than in the controls [ ] . therefore, our data suggests that hbov are not causally associated with gastrointestinal disease in the uk population as a whole, nor in children. the prevalence of detection of hbov in stool samples in previous studies varies widely (see summary in table ), but most coincide in reporting the highest prevalence in children. hbov infections were detected all year round in the uk although a tentative peak was observed in the spring/early summer months in (between april and june). different seasonal patterns in the peak prevalence of hbov have been reported in different countries (see table ), of the hbov positive samples, ( . %) were genotyped in the type-specific assays. hbov- was found predominantly in controls (p, . ) and the prevalence of hbov- was similar in cases and controls. both hbov- and - were predominantly found in children. hbov- was predominantly associated with gastroenteritis cases (p, . ). the overall prevalence in cases was . % and . % in controls, however, in children , year of age, the prevalence in cases and controls was similar, . % and . %, respectively. the prevalence of hbov- in children in the uk was significantly lower than that reported in a study in australia, in which hbov- was detected in . % and . % of the cases and controls, respectively [ ] . the findings of the study in australia lead to the proposal of hbov- as an important aetiological agent of infantile gastroenteritis. it is noteworthy however, that in the australian study, the association of hbov- with gastroenteritis was only significant when cases with a bacterial co-pathogen were included in the analysis. although in the present study hbov- in the absence of other enteric pathogens was found more frequently in cases than in controls, the small numbers found in such large study suggest that the role of these viruses in iid, if any, is likely to be small. a lack of correlation between hbovs or hbov- and paediatric gastroenteritis was also reported in several smaller studies published elsewhere [ , , ] . a total of % of the hbov-positive samples could not be genotyped using the genotype-specific pcr assays. the majority of these untyped samples ( %) had ct values . . this suggests that failure to type may be associated with low viral loads and differences in the relative sensitivities of the genotyping assays compared to the detection assay. although under experimental conditions and using plasmid controls the sensitivities of all assays were comparable, it is likely that when applied to true clinical samples the sensitivity of the type-specific assays was inferior, possibly due to as yet not identified strain variability within genotypes. also, a hbov type -specific assay was not included in this study, therefore, any possible hbov infections would not have been typed. of the panel of samples that were tested in an alternative pan-hbov pcr, the strains typed through sequencing were hbov- ( samples), hbov- ( sample) and hbov- ( samples). furthermore, the distribution of untyped hbovs was not significantly different in cases and controls. hbovs in the absence of other enteric pathogens were seen in % of the hbov-positive samples, and more frequently in the controls, . % vs . % in cases. no significant difference in hbov load was observed between cases and controls, or between the samples positive for hbov alone or in the presence of other pathogens. previous studies have investigated the relationships between viral load and disease severity [ , , , , ] . in respiratory infections significantly higher hbov loads were seen in samples collected from children positive for hbov alone than in those from children with co-infections. in respiratory infections also, viral loads . were associated with disease, whereas loads , were associated with asymptomatic children [ , ] . this lead to the suggestion that higher viral loads are indicative of a causative role of hbov in respiratory infections [ , ] . however, brieu et al [ ] found no significant correlation between viral load and clinical symptoms or disease severity. in conclusion, the results obtained from investigating for the presence of hbov dna in archived dna samples from a large and previously well described case-control study of iid suggest that hbov, including hbov- ,do not appear to be a significant cause of gastroenteritis in the uk population, and particularly in the paediatric population. although hbovs are relatively frequent across all ages, and in particular in preschool age children, they are found just as frequently among children and adults without symptoms of gastroenteritis. cloning of a human parvovirus by molecular screening of respiratory tract samples new dna viruses identified in patients with acute viral infection syndrome epidemiological studies of parvovirus infections in calves on endemically infected properties minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in canadian children in a -year study complete coding sequences and phylogenetic analysis of human bocavirus (hbov) the association of newly identified respiratory viruses with lower respiratory 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children hospitalized for acute gastroenteritis: a case-control study evidence of human coronavirus hku and human bocavirus in australian children human bocavirus in children detection of human bocavirus- in children with acute gastroenteritis in south korea high prevalence of human bocavirus and its role in childhood acute gastroenteritis in china human bocavirus and acute wheezing in children human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants quantitation of group a rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in south india diagnosing rotavirus a associated iid: using elisa to identify a cut-off for real time rt-pcr diagnosing norovirus-associated infectious intestinal disease using viral load human bocavirus infection in children with respiratory tract disease this study was conducted using existing material and data form the iid- study, which was funded by the food standards agency. we would like to thank the iid study executive committee for their support in approving the use of the archive dna material for this study. key: cord- - e xn kj authors: falcón-lezama, jorge abelardo; santos-luna, rené; román-pérez, susana; martínez-vega, ruth aralí; herrera-valdez, marco arieli; kuri-morales, Ángel fernando; adams, ben; kuri-morales, pablo antonio; lópez-cervantes, malaquías; ramos-castañeda, josé title: analysis of spatial mobility in subjects from a dengue endemic urban locality in morelos state, mexico date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: e xn kj introduction: mathematical models and field data suggest that human mobility is an important driver for dengue virus transmission. nonetheless little is known on this matter due the lack of instruments for precise mobility quantification and study design difficulties. materials and methods: we carried out a cohort-nested, case-control study with individuals ( cases, intradomestic controls and population controls) with the goal of describing human mobility patterns of recently dengue virus-infected subjects, and comparing them with those of non-infected subjects living in an urban endemic locality. mobility was quantified using a gps-data logger registering waypoints at -second intervals for a minimum of natural days. results: although absolute displacement was highly biased towards the intradomestic and peridomestic areas, occasional displacements exceeding a -km radius from the center of the studied locality were recorded for all three study groups and individual displacements were recorded traveling across six states from central mexico. additionally, cases had a larger number of visits out of the municipality´s administrative limits when compared to intradomestic controls (cases: . versus intradomestic controls: . , p = . ). we were able to identify extradomestic places within and out of the locality that were independently visited by apparently non-related infected subjects, consistent with houses, working and leisure places. conclusions: results of this study show that human mobility in a small urban setting exceeded that considered by local health authority’s administrative limits, and was different between recently infected and non-infected subjects living in the same household. these observations provide important insights about the role that human mobility may have in dengue virus transmission and persistence across endemic geographic areas that need to be taken into account when planning preventive and control measures. finally, these results are a valuable reference when setting the parameters for future mathematical modeling studies. a a a a a dengue fever (df) is the most important arthropod-borne viral disease in the world. it is caused by infection with any of the four dengue virus (denv) serotypes. nearly half of the human population inhabits areas with denv transmission. in mexico dengue incidence and severe cases have been increasing in the last decade. to date, there is no specific treatment or vaccine for df and vector control stands as the cornerstone for df prevention [ , ] . df is an important public health problem, especially in urban areas [ ] , where it usually presents in large outbreak. the costs of treatment and management during a df outbreak are a serious burden for health systems, especially when there is a risk for saturation of health facilities [ ] . the actors that are necessary for denv transmission are fairly well understood. nonetheless, some of the dynamical features about these actors still need to be elucidated in order to understand how they impact on transmission. human mobility has been studied in relation to other infectious diseases, where its role as an important driver for disease transmission has been proven [ , ] . mathematical models have suggested that local scale human mobility may play a role in denv transmission, outbreak persistence, and control efficiency [ , ] . however, little information is available on detailed human mobility patterns in geographic areas where df is endemic or on confirmed cases during an outbreak. recently, gps-based technologies have been tested and shown to be a reliable and acceptable tool for quantifying human mobility [ , ] . human mobility has been described in relatively recent reports by using indirect measures [ , ] . for these reasons, we studied the micro and macromobility of dengue virus-infected subjects in an endemic locality. here we present the results of a cohort-nested case-control study on a dengue endemic urban locality in mexico. the protocol of the project was reviewed and approved by the comité de etica y de prevención de conflictos de interes (institutional review board) ci no and departamento de investigación (external review) servicios de salud de morelos, mexico dei/cei/ / . cohort-nested, case-control study. sample: individuals ( cases, intradomestic controls and population controls) with age older than , and residents in axochiapan, morelos state, méxico, were selected from the cohort "peridomestic infection as determinant for dengue virus transmission" [ ] . they were assigned into three study groups: a. cases were individuals with laboratory evidence of recent, symptomatic or asymptomatic, denv infection, and identified as the only persons infected within their households during the study. b. intradomestic controls, were individuals with a negative serological result for recent denv infection, living in the same household with a case; c. population controls were individuals with a negative serological result for recent denv infection, randomly selected from the same locality. all controls tested negative for denv during the same period and using the same validated techniques than cases (igm or igg capture elisa) as reported in the cohort study [ ] . partici-pant´s selection was performed as follows: cases were approached first, if accepted participation an intradomestic control was randomly assigned from the pool of subjects living in the same house that had both baseline and final negative elisa results. for each pair of in-house participants, a randomly selected population control was then assigned (fig ) . given the limited number of available gps loggers and the three-month time frame for the follow-up, the recruitment was limited to a maximum of cases with their respective intradomestic and population controls. between may and september, , with prior signed informed consent, all participants were provided with a portable gps (gps data-logger, transystems mod. a+), programmed for recording its position at -second intervals (variables date, time, latitude, longitude, altitude and speed), during hours a day, for a minimum period of days. participants were instructed to carry their gps at all times whenever they left their homes and to recharge the equipment's battery daily during their in-home resting times. this follow up was performed in cases identified during the immediate previous season, on average one year after diagnosis confirmation, in order to match the activities performed during the high transmission season, and under the assumption that their mobility patterns remained unchanged after disease, and constant through time. a web-based interface was developed to import the text files from gps equipment to a main database. variables were homogenized, waypoints in the initial and final days of each individual gps track (comprising incomplete days) were eliminated in order to standardize the period of time to be analyzed starting at : : hours on the second day of follow up and ending at : : on the last to final day of follow up. data were converted into a feature dataset and projected from the geographic coordinate system to a lambert coordinate system (from hexadecimal to metric units) with the purpose of performing arithmetic operations for distance calculations. origin or routinely residence sites were identified by means of an iterative algorithm (mean center) employing waypoints from : : to : : hours, monday to friday. routine residence coordinates were added to the database. distance to home variable (dhome) was calculated for each extradomestic waypoint using sql applying the following formula: where; x = xhome (x coordinate from home), x = xccl (x coordinate from each waypoint), y = yhome (y coordinate from home) y, y = yccl (y coordinate from each waypoint). waypoints within the peridomestic area (dhome < m) were identified. distance, speed, altitude and time differentials were created: where: xccl = (x coordinate from previous waypoint), xccl = (x coordinate from current waypoint), yccl = (y coordinate from previous waypoint) and, yccl = (y coordinate from current point) displacement and spatial permanency variables for each subject with complete data were generated. visit sites were defined as those areas out of the individual's home with a m radius in which each participant remained static for a period enough to allow a potential effective interaction with local vectors. these sites were identified by generating an algorithm through which visit clusters were formed using the following criteria: stops lasting minutes or longer, a distance from home of m or farther, distance of the current waypoint from previous waypoint < m, and speed for the current waypoint of km / h or less. for each cluster (visit site) a centroid was calculated. common visit sites for cases were identified as hexagonal cells with m radius [ ] which were visited by at least two different cases at a given time, and where the proportion of different visiting cases was at least two thirds of the total visiting population for that cell. common visit sites for controls were identified as hexagonal cells with m radius which were visited by at least two members from each control population and not visited by any of the cases. the geographic universe in the study was divided in five areas ( .-inside the house, .-out of the house but in the locality, .-out of the locality but in the municipality, .-out of the municipality but in the state, .-out of the state), limited by four buffers. a circular buffer with m radius around each participant's home limited the first area and three additional polygonal buffers were drawn according to the administrative limits for the locality, municipality and state. arcgis arcinfo was used for processing and analyzing spatial data, sql server was used to create a geodatabase, arc sde was used as interpreter between entre sql and arcgis. statistical analyses were performed using stata . fifty randomly selected cases were asked to participate in the study from which ( %) accepted participation. all approached controls agreed to participate. in total individuals ( cases, intradomestic controls and population controls) were recruited. our drop-out rate was lower than % ( / ) since one participant (intradomestic control) did not finish the follow-up due to the loss of the assigned gps logger. table describes the main characteristics of the subjects in each group. no statistically significant differences were observed in most of variables except in age, since cases were significantly younger than the intradomestic or population controls (cases mean: . , sd: . ; intradomestic controls mean: . sd: . ; population controls mean: . sd: . . p = . ). of participants, ( . %) participants completed their follow up since one gps used by an intradomestic control went missing. the final database contains , , waypoints from these participants, and all participants were followed by a mean of . continuous days. as for the number of days of follow-up for each group no differences were recorded. as expected, most of the waypoints in the population fell within the intradomestic area (< m radius from home centroid). as distance from home (absolute displacement) increased, we observed a marked decrease in the proportion of waypoints. all three groups presented a small peak when the distance reached the m radius. from this point the proportion of waypoints quickly decayed (fig ) . no differences were noticed for absolute displacement among the groups. the hourly distribution of recorded waypoints out of the participant's homes is shown in fig . as expected, participants usually left their homes early in the morning and returned by the end of the day. although we recorded waypoints out of the participants' homes in every hour of the day, the period comprised between : pm and : pm registered the peak in the number of waypoints recorded out of the homes, and this number decreased steadily as the day progresses. the pattern during weekdays ( fig a) suggests that cases leave their homes and return to them slightly earlier than control groups. as for the weekends (fig b) , both control groups show a similar pattern to that observed for weekdays, nonetheless, cases seem to remain in their homes more often and return earlier. table shows values for different mobility variables. no significant differences were recorded among groups for the following variables: mean distance from home at all times, maximum recorded distance at any given time, and mean time spent in each geographic area at any speed or at static speed. nonetheless, when comparing the number of visits per geographic area, the cases had fewer recorded visits in the area out of the locality but in the municipality ( . vs . , p = . ), and more visits in the area out of the municipality ( . vs . , p = . ), both compared to intradomestic controls. these differences were statistically significant. consistent with this behavior, although non-statistically significant, cases visited more states, municipalities, and regions with high dengue incidence through their follow up, in comparison to both control groups. we next examined the proportion of waypoints recorded by the comparison groups in each area stratified by age (fig ) . there is a notorious difference in the proportion of waypoints among the cases, observing an increase of nearly percentile points in the intradomestic waypoints, recording the highest frequency in cases under age (fig a. however the difference not statistically significant (cases < : median . %, interquartile-range . - . ; cases ! : % iqr - . ; p = . ). we also observed a difference in the area out of the municipality but in the state, where the group of cases aged and older spent the highest proportion of time (age < : % iqr - %; age ! : . % iqr - . ; p = . ). there was no significant difference between cases and population controls, regardless of age. when comparing cases versus intradomestic controls, we observed a statistically significant difference in time spent in area out of the municipality but in the state (cases: . % iqr - . ; ic: % iqr: - . ; p = . ). we found differences in mobility patterns when analyzing data by gender ( fig b) . women had a higher proportion of waypoints within the intradomestic area than men. these differences were statistically significant for intradomestic area (male: . % iqr: . - . ; female: . % iqr . - . ; p = . ), out of their homes but in the locality (male: . % iqr . - ; female: . % iqr . - . ; p = . ) and the area out of the locality but in the municipality (male: . % iqr: . - . ; female: . % iqr: - . ; p< . ). nonetheless, linear mean (p = . ) and maximum (p = . ) distances were not. a) cases vs. intradomestic controls. a conditional logistic regression analysis was performed, including variables identified in the bivariate analysis as having a p value < . , and by data mining techniques using all variables as reported previously [ ] . for bivariate analysis variables were age (continuous and dichotomic [under or and older]) gender, occupation (intra or extradomestic), education (dichotomic), and the proportion of time spent in each geographic area. for the data mining we considered the whole data base. the final model included age (or: . ic % . - . ; p = . ) and the area out of the municipality but in the state (or . ic % . - . ; p = . ). we observed a protective effect in the and older group, and a risk effect when the proportion of time spent in the area out of the municipality but in the state is increased. b) cases vs. population controls. a multiple logistic regression analysis was performed including variables identified with p value < . in the bivariate analysis and data mining techniques using all variables as reported previously [ ] . for bivariate analysis, only the variables age (continuous and dichotomic [under or and older]), gender, occupation (intra and extradomestic), education (dichotomic), proportion of time spent in each area and linear distance were taken into consideration. for the data mining we considered the whole database. the final model included: occupation (or . ic % . - . ; p = . ), proportion of time in the area out of the municipality but in the state ( . ic % . - . ; p = . ), proportion of time in the area out of the locality but in the municipality (or . ic % . - . ; p = . ), and age (or . ic % . - . , p = . ). next we analyzed the geographic distribution of the recorded waypoints for each group, both locally and regionally (fig ) . all three groups recorded waypoints exceeding a km radius from their homes (fig a, b and c ). as expected, most of recorded waypoints were located within the locality. nonetheless, all three groups recorded waypoints exceeding the locality, municipality and state limits. these trajectories were headed mainly to the east, north and west of the locality, and were consistent with the location of the main cities in the area, including cuernavaca (population , ) and cuautla (population , ), the capital city and the second most important city in the state of morelos, respectively. the geographic distribution of the visits performed by cases and df cumulative incidence for the central mexico region, during year , is shown in fig . as seen, this group performed visits to locations with and without df transmission. the number of states, municipalities and regions with high dengue incidence is shown in table . within the locality of axochiapan the most visited areas were identified by dividing the locality in m-radius hexagonal cells (fig a) . the most visited cells were those located in the locality's central area, which correspond to the location of the main market, road junctions and main administrative and / or service offices. the location of the cells considered as common visit sites for cases are shown in fig b. unlike in fig a, the geographical distribution of these cells tends to be peripheral with respect to the locality. using google earth™ we identified the geographic features of each of the cells that was classified as a common visit site for cases. fourteen out of fifteen cells were geographically located within the locality of axochiapan, morelos, and the last one was in the central area of a neighboring small locality (town of tzicatlán) in the state of puebla. as for their typology, xix out of cells clearly corresponded to residential areas (including that in the neighboring state), one cell was a residential area adjacent to a local large business, four cells included small processing plants, a warehouse and a local business, three peripheral cells were crop fields and one cell was clearly a soccer field. previous works have used gps tools for measuring exposure to infectious diseases [ ] [ ] [ ] . in df, recent works in the endemic area of iquitos, peru, have elegantly described human population mobility [ , ] . however, few data are available for infected cases so far. our work builds upon our knowledge of the role played by mobility in denv transmission documenting spatial mobility of subjects from an endemic region that had, or had not been recently infected by denv. our data show that the people from axochiapan stay within their houses or surrounding areas most of the time. this is consistent with previous observations in iquitos, where population rarely moves more than km away from their homes [ ] , however, some individuals recorded movements to very distant locations through the relatively short follow-up period. these movements were present in all three study groups and exceeded a -km radius from the center of the study, covering the neighboring states of morelos, state of mexico, puebla, mexico city, tlaxcala and hidalgo. all of these states are located in the mexican central plateau, which is also the best connected region in the country and therefore it is not difficult to reach those destinations by commute travel [ ] . surprisingly, spatial mobility in humans was not geographically symmetrical in our study, since no movements were recorded to state of guerrero, which is a coastal, highly endemic area for dengue and also a popular destination for leisure activities. as for the reason why the mobility of the individuals is biased towards central plains in mexico and practically absent towards the southern regions, it was a surprising finding also for us, but we think that it has to do with two factors: first the economic activities in axochiapan are mainly related to agriculture, trade and services which are strongly influenced by the needs of mexico city and its metropolitan area, comprised also by the states of morelos, puebla, méxico and hidalgo. the main cities of these states were those that were visited by the cases and in a lesser extent by the controls. secondly, we did not perform any follow-up during summer and christmas holidays, which in mexico are specific periods for leisure. these activities are usually performed in places that might be different that those observed in our study, including the beaches in the southern coast. it is possible that had we performed our follow-up in vacation periods the observed mobility might have been different, and also leave us with a very interesting research question for the future. the large size of the area covered by these few individuals from a small locality (axochiapan has roughly , inhabitants) is of capital importance, given the fact that in mexico, and probably in many other places, epidemiological surveillance, prevention and control activities for df are mainly planned, supported and executed by local health authorities, who rely on the information generated by a number of systems, most of them automated [ ] , but that are usually restricted to their local administrative limits, namely municipality, sanitary jurisdiction or state at best. thus when df outbreaks overcome those limits and a wider coordination is needed, it is probably that the outbreaks are already established and the window of time for effectively applying control measures has been lost. our data show that the cases group had the largest difference on the time spent in the home area with strong age dependence. older cases spent less time in their homes compared to younger cases. as far as the number of visits is concerned, subjects in the cases group, especially those aged over , performed many and more distant visits, than subjects in the intradomestic control group. this difference with the population control group was less marked. this scenario suggests that the population aged over might play an important role in denv persistence and dispersion perhaps working as geographic spreaders. our group previously determined dengue incidence for this age group and proposed a dynamic model which seems to be corroborated by the results presented here [ ] . infected individuals, both symptomatic but also asymptomatic [ ] , may facilitate the infection of extradomestic mosquito populations in a local scale as models have suggested [ ] , or at a regional scale introducing or exporting the virus. given the fact that we performed an uninterrupted -hour follow up, we were capable to register the time when individuals left their homes for whatever activity they performed. to our surprise, we recorded waypoints out of the participant's homes virtually at any hour of the day. although the majority of records show that people in axochiapan have a day-light pattern of activities, we recorded waypoints from cases, between : and : am, which were consistent with participants' declared jobs, which were related to nocturnal activities such as bakers and workers from the local stone processing plant. the dispersion patterns described above, both in space and time, might be of importance in the results obtained in the control of denv transmission in this and similar small localities. the usual schedule considered by local health authorities for applying preventive measures, which favors early hours for insecticide spraying and the visits by entomological control brigades, in a geographically focalized strategy might hinder the efficacy of the actions by the mere fact that people moves from their homes and remain away during the time these actions are normally applied. in our data, the hourly pattern for activity suggests that cases might leave earlier their homes during weekdays, and thus their homes might have a higher probability to remain closed by the time health authorities apply preventive or control measures. it is important to notice the high mean age of the cases in axochiapan, in both the participants in the study and those recorded historically in the state of morelos, in comparison to other endemic areas from mexico and the americas. this is however, consistent with a previous work in the area [ ] , and is probably due to the fact that most ( out of members of the cases group) of the cases that we studied were asymptomatic, also, although not statistically significant, the mean age of the asymptomatic individuals was higher than that from symptomatic individuals (mean age asymptomatic: . vs mean age symptomatic: . , t test p = . ). thus, suggesting a possible stronger role of asymptomatic population with age above as spreaders for denv transmission. previous studies have described that visiting other cases' households is a risk factor for denv dispersion [ ] ; working sites have also been suggested as possible transmission sources outside of cases' households [ ] . models have shown that sites outside homes can play a role in denv transmission and infection [ ] , and a recently published work showed positive correlations in thailand between the aedes spp. house index and specific landscape features [ ] . our findings are consistent with these data since cases coincided in houses different to their own in at least five different geographical locations. additionally we found cases that coincided in four potential working places, and a soccer field. as far as we know, this is the first time that leisure sites have been documented as possible areas for denv transmission. the lack of study of such sites has previously been pointed out as a weakness in the study of human mobility [ ] . as for the common visit sites for controls, we identified cells that were visited only by individuals from both control groups but not by cases. these cells included only one potential working site; whereas the cells commonly visited by cases included several likely workplaces. the identification and study of the extradomestic sites where people coincide is relevant: a recent simulation model has concluded that the selection of areas for df control out of the cases' homes is important not only in terms of the time the subjects spend in them, but also because of the local vector:host ratio, and the other habitual destinations of people that visit the same area [ ] . it is possible that much of the movement in a given society is driven by specific population needs and the possibility to fulfill them within or outside from their own locality. axochiapan is a fairly small and well connected locality to other small cities, all of them endemic for df. it is not unrealistic to think that some of the population needs can be readily fulfilled within the same locality whereas other cannot, compelling the population to move away in a permanent or transitory fashion. if a specific population such as that with age older than becomes infected and effectively play a role as spreaders, then perhaps small and peripheral localities to larger cities might have a key importance in sustaining denv transmission across large geographic areas. the assessment of such situations requires a critical review of existing data and the generation of specific studies that may help us to recast current models and more importantly, to completely understand urban denv transmission [ ] . we have identified some weaknesses in our study. the most relevant is our small sample size which might have hindered our capability to identify clear patterns in the mobility from this mexican community. there is a possibility that any individual belonging to either control group might have get infected during the follow up; thus making her/him eligible to become a case, therefore disqualifying him/her for being a suitable control and consequently introducing an information bias; nonetheless, we believe that, although a possibility, this was negligible due to two reasons: first, the follow-up of days was very short for this event to occur, and secondly because while recovering each gps, we asked all individuals whether they had experienced fever or any other symptom suggesting dengue infection during the follow-up. none of the participants reported any change in their health status. although we understand that a more robust argument to ensure the infected/uninfected status might be performing an elisa to each control after finishing their follow-up in order to be certain about their exposure, financial constrains made impossible this procedure. possible future improvements in our study are: increase the limited sample size, the inclusion of adequate representation for the population under the age of , which probably is a relevant group for transmission during the initial and focalized phases of a df outbreak. although no schools could not be identified in our study as a common site visited by cases, this observation needs to be taken cautiously since our study did not consider the follow-up of children usually studying elementary education. thus, we cannot rule out any role of these sites in dengue transmission at younger ages. additionally, extending the duration of the follow up might improve the chances of successful identification of patterns whose frequencies are longer than a week, such as wage collection, bill payments, and communitarian meetings, among others. this last topic is essential; nonetheless it is limited by technical issues that might be addressed as technology for massive and continuous long-lasting follow up becomes available. finally, the main reasons for which the participants move were not deeply explored in our study, thus we cannot be certain whether the recorded movements indeed depend on non-satisfied needs or on leisure activities. we can only assume that at least those movements performed during the mornings and afternoons between monday and friday correspond to real needs such as employment, education, and supply acquisition, and those performed during weekends are related to leisure. some causes for loss of gps information in field studies have been recently described [ ] . although some of these causes might be present in our study, we believe they did not represent significant sources of bias or information loss, since we took some specific measures. for example, people were prevented of accidentally turning the gps off by strapping a tape in the controls. in order to diminish the probability that the participants could forget their gps units at home we performed a weekly phone call reminding them the importance of the usage attachment according the protocol during each individual follow-up. barriers to signal were not important in the studied area since it is located in a plateau with few elevations, and buildings taller than -stories are practically absent. finally, the gps equipment used in the study had battery autonomy of up to straight hours and enough memory for recording up to five times the mean number of waypoints programmed to collect in each subject. based on our own data and that from recently published works we conclude that gpsbased technology is a solid tool for the study of detailed human mobility in denv transmission or other infectious diseases, which can and must be adopted in public health and epidemiology as a basic instrument. the important geographic dispersion in our results demonstrates the necessity for studying the potential role that human mobility has in denv transmission and outbreak duration and also a strong argument to study and clarify the role that asymptomatic cases might have in dengue virus dispersion. furthermore our data strongly suggest that the size of the areas considered for prevention and control of df outbreaks needs to be revised and that it is necessary to integrate this knowledge into the planning of preventive and control measures, which usually are prone to using basic shapes such as circles or squares as geographic references in order to define limits, ranges, trajectories and points of origin. it is clear that human populations move normally across geographical areas and not only during holidays or vacations. according to our data, the magnitude of these displacements is larger than that considered as an administrative responsibility for local health services providers. this is relevant for denv transmission if a large fraction of that mobile commuting population is also asymptomatic but viremic, facilitating with their movements the exposure of local uninfected mosquito populations with the virus, which might result in an increased geographical dispersion and persistence of the outbreaks due a continuous process of spreading and reintroduction of the virus to susceptible populations. finally, we believe that the data here reported should be valuable for parameterization of mathematical models exploring specific issues in dengue epidemiology such as geographical dispersion of human activities, contact rate among humans in intermediate spots, optimal range for vector control coverage, optimal target places for health promotion activities, impact of coordinated regional collaboration, and transmission dynamics among satellite and large cities. all essential topics that are still to be understood and weighed as drivers in the transmission of this and other mosquito-transmitted diseases. dengue and dengue heamorrhagic fever world health organization. dengue guidelines for diagnosis treatment, prevention and control urbanisation and infectious diseases in a globalised world the global economic burden of dengue: a systematic analysis travel 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water-contact patterns associated with schistosomiasis distribution and interspecies contact of feral swine and cattle on rangeland in south texas: implications for disease transmission health &demographic surveillance system profile: the kombewa health and demographic surveillance system (kombewa hdss) multiple outbreaks for the same pandemic: local transportation and social distancing explain the different "waves" of a-h n pdm cases observed in méxico during asymptomatic humans transmit dengue virus to mosquitoes house-to-house human movement drives denguevirus transmission epidemiology of dengue and dengue haemorrhagic fever in a cohort of adults living in bandung analyzing the spatio-temporal relationship between dengue vector larval density and land-use using factor analysis and spatial ring mapping population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and noncommuting dengue cases recasting the theory of mosquito-borne pathogen transmission dynamics and control strengths and weaknesses of global positioning system (gps) data-loggers and semi-structured interviews for capturing fine-scale human mobility: findings from iquitos authors wish to thank to servicios de salud de morelos for its support to this project. the authors have declare that no competing interests exist. key: cord- -wtestt i authors: jung, eunok; de los reyes v, aurelio a.; pumares, kurt jan a.; kim, yangjin title: strategies in regulating glioblastoma signaling pathways and anti-invasion therapy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wtestt i glioblastoma multiforme is one of the most invasive type of glial tumors, which rapidly grows and commonly spreads into nearby brain tissue. it is a devastating brain cancer that often results in death within approximately to months after diagnosis. in this work, optimal control theory was applied to regulate intracellular signaling pathways of mir- –ampk–mtor–cell cycle dynamics via glucose and drug intravenous administration infusions. glucose level is controlled to activate mir- in the up-stream pathway of the model. a potential drug blocking the inhibitory pathway of mtor by ampk complex is incorporated to explore regulation of the down-stream pathway to the cell cycle. both mir- and mtor levels are up-regulated inducing cell proliferation and reducing invasion in the neighboring tissues. concomitant and alternating glucose and drug infusions are explored under various circumstances to predict best clinical outcomes with least administration costs. glioblastoma multiforme (gbm) is the most common and the most aggressive type of brain cancer. the median length of survival time is approximately to months following diagnosis. gbm is characterized by anaplasia, nuclear atypia, cellular pleomorphism, mitotic activity, and more importantly, alternating phases of rapid proliferation and aggressive invasion into the surrounding brain tissue. this leads to an inevitably critical recurrence even after the surgical resection of the main tumor mass [ , ] . the mainstay of treatment for gbm is surgery, followed by radiotherapy and chemotherapy. despite advances in these approaches, glioma cells can still invade the neighboring tissues beyond detection leading to tumor recurrence. high probability of main treatment failure also encourages researchers to investigate the use of innovative treatments when the first line of therapy has failed, in order to improve clinical outcomes [ ] . in the tumor microenvironment (tme), glioma cells encounter many challenges including hypoxia, acidity, and limited nutrient availability. to maintain rapid growth, tumor cells need to adapt to these biochemical changes and modify their metabolic activity by increasing glycolysis even in the presence of oxygen. this process is called the warburg effect which requires consuming considerable amounts of glucose [ ] . the tricarboxylic acid cycle, or krebs cycle, plays an important role in the breakdown of organic fuel molecules and the survival in non-a a a a a hypoxic normal differentiated cells. these molecules include glucose, fatty acids, and some amino acids. while differentiated cells favor this type of metabolism, which is very efficient in terms of atp production, tumor cells adopt the inefficient aerobic glycolysis producing relatively large amounts of waste product in the form of lactic acid [ ] . this may provide cancer cells the advantage of not having to depend on oxygen as an energy source especially in a hostile tumor microenvironment, thus leading to longer survival [ ] . inhibition of glycolysis may also prevent drug resistance thus a better understanding of this metabolic pathway may lead to better treatment options and clinical outcomes [ ] . developing strategies of metabolic adaptation, angiogenesis, and migration is critical for cancer cells in order to survive metabolic stress and ensure enough nutrient supply as tumor mass accumulates where glucose supply may fluctuate due to heterogeneous biochemical and biophysical conditions [ ] . therefore, adequate cellular responses to glucose withdrawal are critical for cancer cell survival. cancer cells then activate the -adenosine monophosphate activated protein kinase (ampk) pathway under metabolic stress. it is the master cellular sensor of energy availability which enhances glucose uptake and conserve energy, thus avoiding cell death [ ] . micrornas, also abbreviated as mirna, are approximately nucleotide single-stranded non-coding ribonucleic acids (rnas) that are known to regulate gene expression [ ] . dysregulation of microrna expression has been linked to oncogenic and tumor suppressor activities in several types of cancer, including gbm [ , ] , where altered mirna expression contributes to tumorigenesis [ , ] . godlewski et al. [ ] identified an interesting mechanism of glioma cell migration and proliferation wherein a particular microrna, mir- , and its counterpart, ampk complex (cab / lkb /strad/ampk), determine whether the cell favors growth at the expense of invasion or conversely. moreover, they also identified a potential feedback loop between lkb and mir- allowing for a sustained and robust response to glucose withdrawal [ ] . it was found out that (i) under high (normal) glucose conditions, up-regulation of mir- leads to the down-regulation of ampk complex, which then leads to elevated proliferation and decreased migration of glioma cells and (ii) glucose withdrawal induces down-regulation of mir- and up-regulation of ampk, which promotes cell migration with reduced proliferation. the mathematical models developed by kim et al. [ , , ] describe the effects of the mir- -ampk core control system on cell proliferation and migration in glioblastoma. it explains the response of mir- to high and low glucose levels as well as the mutual antagonism between mir- and ampk complex concentrations. kim et al. [ ] then extended the model to include the dynamics of mammalian target of rapamycin (mtor), which is a protein kinase that links with other proteins as well as to the cell cycle dynamics, to form the mir- -ampk-mtor core control system. this mutual antagonism between mir- and ampk, which was predicted in these mathematical models [ , [ ] [ ] [ ] , was confirmed by recent experiments. for example, ansari et al. [ ] recently found that (i) the mir- transcription in gbm cells is induced by unrestricted activity of its transcription factor oct (official gene symbol pou f ) in the presence of abundant glucose, resulting in ampk inhibition through direct targeting of cab in the lkb complex; and (ii) the mir- level is inhibited through the phosphorylation and inactivation of oct at s by ampk in response to glucose depletion-induced metabolic stress, leading to a reciprocal negative feedback loop between mir- and ampk. in this case, suppression of mir- in turn leads to sustained ampk activities and a robust response to glucose withdrawal in gbm cells. the multiscale mathematical models [ , [ ] [ ] [ ] also predict the growth-invasion cycling patterns of glioma cells in response to fluctuating glucose uptake in the tumor microenvironment. the core control system predicts bistability and hysteresis bifurcation when delayed down-regulation of mir- activities along certain molecular pathways would induce glioma cells to stay longer in the proliferative phase despite relatively low glucose concentrations, making this mechanism a therapeutic target. the cell cycle represents an integrated series of events that regulates complex processes including cell proliferation, cell division and dna replication, regulated by a complex hierarchy of genetic and metabolic networks which involves several transition states of varied lengths and checkpoints [ ] . the stages of the cell cycle are as follows: (i) synthesis phase (s), a period where dna replication occurs; (ii) gap phase (g ), during which proteins required for mitosis are produced; (iii) mitosis phase (m), a period where chromatin condensation, nuclear envelope breakdown (nebd), chromatid separation, and cytokinesis happens; (iv) gap phase (g ), in which genes necessary for dna replication are activated and the protein agents of s phase progression are accumulated; and (v) resting phase (g ), a state in which cells can exit the cell cycle and enter a phase of quiescence or relative inactivity [ ] . the progression of mammalian cell cycle is tightly regulated by coordinated activation of cyclin-dependent kinases (cdks) family [ ] . the cdks are positively regulated by cyclins and negatively by cdk inhibitors (cdkis) such as the proteins p , p , p and p . in cancer cells, cyclins are over-expressed while cdkis are under-expressed which results in the dysregulation of the cell cycle, and promoting uncontrolled cell growth [ ] . tyson and novak [ , ] identified that the transition between two stable steady states, g and s-g -m cell-cycle phases, are described using the kinetic relations of the model that is controlled by changes in cell mass. a standard gbm treatment is surgery followed by chemotherapy and radiotherapy. however, even under best circumstances, the mean survival of this disease is about a year. poor outcomes of standard care treatments are due to the topographically diffuse nature of the disease [ ] . by the time of diagnosis, typical gbm cells may have widely spread throughout the brain tissue [ ] [ ] [ ] , increasing the potential of recurrence. thus, annihilation of distant tumor satellites is implausible despite surgically removing all the essential tumor seen on enhanced mri scan [ ] . knowing the exact margins of a tumor mass in real patients is indeed a daunting task. in this study, it is assumed that a major tumour mass has been surgically removed and that the infiltrative tumor cells are near the surgical site. the objective is to prevent the glioma cells from further diffusing into the surrounding brain tissue. localization approach will be utilized, that is, glioma cell invasion will be blocked keeping them in a proliferative phase while also attempting to limit excessive growth before a second surgery [ ] . our analytical tool is primarily based on the framework of optimal control theory, which has been successfully used to make informed decisions involving biological models such as optimal treatment strategies in human immunodeficiency virus (hiv) models [ ] [ ] [ ] [ ] , tuberculosis [ ] [ ] [ ] , and cardiopulmonary resuscitation (cpr) techniques [ , ] . optimal control theory is also applied to the mir- -ampk core control system to determine the intravenous glucose and/or drug infusion protocols with least possible cost under various circumstances in de los reyes, et al. [ ] . recently, kim et al. [ ] developed an intracellular signaling pathway model that extends the mir- -ampk-mtor core control system including the cell cycle dynamics. in this work, a potential control problem is formulated in order to maintain high levels of mir- and mtor (low levels of ampk) inducing cell proliferation prohibiting cell motility and invasion to the neighboring tissues. with glucose levels as a key regulator of mir- activity which also activates mtor in the downstream signaling pathway, glucose intravenous infusion is considered to up-regulate mir- and mtor concentrations above certain threshold values. in addition, a drug suppressing the inhibitory effect of mtor by the ampk complex is incorporated. this drug can be administered concomitantly or alternately with glucose as a secondary intravenous infusion. the controls are then given by dose rates of glucose and drug intravenous administrations regulating upstream and downstream signaling pathway, respectively. solution of the optimal control problem aims to determine infusion protocols with minimal glucose and drug amount, and least administration costs. hence, glucose and drug levels are regulated to prevent rapid tumour growth, hyperglycemia, and further drug complications. the results propose plausible glucose and drug intravenous infusion controls which indicate the time of administration, frequency, number of administrations, and dosages of glucose and drug per infusion. in the current study, we will first present the mir- -ampk-mtor-cell cycle intracellular signalling pathway developed by kim et al. [ ] . a drug module is incorporated to up-regulate mtor activities inducing cell proliferation. then an optimal control problem is formulated with the goal of activating mir- and mtor levels through glucose and drug intravenous infusions. two different infusion protocols will be explored, namely, concomitant and alternating glucose and drug intravenous administrations. optimal solutions for two strategies are presented and results on frequency, dosage per infusion, total glucose and drug amount, and relative cost incurred in the administrations are compared. the conclusion section discusses and summarizes the optimal control results, and provides outlook for future research directions. in this section, we present the basic components of intracellular signalling pathway of tumor cell containing the core control mir- -ampk-mtor and cell cycle pathway developed in kim et al. [ ] , incorporating a drug which blocks the inhibitory pathway of mtor by ampk complex. the core control model identifies a key mechanism which determines the molecular switches between the proliferative and migratory phases in response to fluctuating glucose and drug levels. the simplified signaling pathways consists of five key determinants of the intracellular structure, namely, glucose level g, mir- level m, ampk complex activity a, mtor concentration r, and drug level d. the intracellular cell cycle dynamics are developed by tyson and novak [ , ] including only the essential interactions for its regulation and control. the model captures the kinetics of chemical processes within the cell such as production, destruction, and different molecule interactions. the transition between two main steady states, g and s-g -m phases, of the cell cycle are described using the kinetic relations of the model that is controlled by changes in cell mass. . also included are the effects of the changes in oxygen dynamics at the macroscopic level through the activation and inactivation of hif- α. this results in changes in cell cycle length. in addition, the cell cycle inhibitory effect of p or p genes of hif- α is incorporated. kim et al. [ ] proposed to link both the mir- -ampk-mtor control system and the cell cycle dynamics to provide a mechanism driving the cell cycle to undergo the quiescent stage g -phase depending on the concentration level of mtor. fig a depicts the detailed schematic diagram of the intracellular signalling networks including mir- , ampk complex, mtor, and key players in the cell cycle module (cycb, cdh , p cdct, p cdca, plk ). kinetic interpretation of arrows and hammerheads represent induction and inhibition in the signaling network, respectively. the dimensionless diagram of the core control mir- , ampk complex, and mtor linking to the cell cycle is depicted in fig b. it should be noted that u g and u d are the sources of glucose and drug with decay rates μ g and μ d , respectively, which can be controlled exogenously. s and s are signaling sources to ampk complex and mtor, respectively, while α, β and γ are inhibition strengths, and ϕ denotes decay. it has been shown that high (normal) glucose concentration yields over expression of mir- levels (down-regulation of ampk complex and up-regulation of mtor) leading to elevated cell proliferation and reduced migration, while low glucose levels leads to down-regulation of mir- activities (up-regulation of ampk complex and down-regulation of mtor) reducing cell proliferation and inducing migration into the surrounding brain tissue [ , , , ] . the effect of various glucose levels in the regulation of the core control is depicted in fig . kim et al. [ ] developed a core control system of glioma cell migration and proliferation by using a regulatory network of key molecules (mir- (m), ampk (a)) as follows: as observed in experiments [ , , ], the regulatory system in eq ( ) includes a mutually antagonistic loop between mir- and ampk complex in response to high and low glucose levels (g). this genetic toggle switch induces a monostable and bistable system, characterizing proliferation and critical cell infiltration of gbm cells in brain [ ] . in the follow-up studies [ , [ ] [ ] [ ] including mtor (r) and cell cycle modules, this mutual antagonism and bistable system played a critical role in developing anti-invasion strategies. a mutually antagonistic feedback loop has been well-studied for its bistable properties by use of mathematical models ([ - ] and other references in [ ] ). for instance, in a study on a genetic toggle switch in escherichia coli [ ], a mutually inhibitory loop between repressor and repressor was in this study, we consider a drug d suppressing the inhibitory effect of mtor by ampk complex where the inhibition strength is given by z(d) = e −d . when d is large, γe −d is small which makes dr/dt to be large. thus, the presence of drug up-regulates mtor activity that could eventually lead to cell proliferation. fig illustrates the mtor bifurcation curve. observe that increasing drug concentration shifts the hysteresis curve upwards keeping the same bistability window. hence, with higher drug levels for the same glucose concentrations, mtor is activated prompting elevated cell growth. in the mir- -ampk-mtor core control system developed by kim and colleagues [ , [ ] [ ] [ ] ] , the bistability regime of main variables emerges in response to glucose levels. as it was shown in [ ], the existence and size of the bistability window (|w b |) depend on other essential parameters and may disappear under perturbations of parameters. as it will be shown later (figs and ), some of key parameters are sensitive in creation or destroying the bistability while other parameters are not. after achieving the equilibrium, continuation of the curve is computed by varying the glucose concentration g and bifurcation points are detected labeled lp and cp for limit and cusp points, respectively. fig a illustrates the hysteresis diagram (g, r)−curves for different s values. in order to obtain the cusp point (cp), fold continuation is computed starting at a limit point where two parameters g and s are activated resulting to codim bifurcations. both parameters (g, s ) are varied along the curve where each point is a limit point for the equilibrium curve at the corresponding value of s . this is depicted in fig b. the cusp point gives the threshold values th m , th a , and th r . a matlab software matcont was used for numerical continuation and bifurcation study of continuous and discrete dynamical systems [ ] . the governing model equations for the dimensionless intracellular signaling dynamics are then described by the following ordinary differential equations d½plk � dt ¼ k ½mass s �½cycb�ð À ½plk �Þ À k ½plk �; strategies in regulating glioblastoma signaling pathways and anti-invasion therapy the cell cycle dynamics and the regulatory core control system are linked by a variable called pseudo-mass ([mass s ]) given by the oxygen dynamics through hif- α is described as and the growth rate μ is expressed as wherem is the probability density function with uniform distribution between − and . this growth rate formulation introduces cell cycle heterogeneity of length between and hrs to account for the natural variability between cell growth rates and to have a non-synchronous population [ ] . the model parameters in system ( ) are listed in tables and . in the current model formulation, mtor (r) is activated/inactivated in the same way as mir- (m). in addition, r links the core control system and the cell cycle dynamics via the pseudo-mass ([mass s ]) which influences the cell mass [mass] and the intracellular proteins (refer to eq ( )). therefore, when glucose supply is high, r is up-regulated (proliferative phase; up-regulated m, down-regulated a) and [mass s ] � [mass] yielding a typical cell cycle (see fig ) . on the other hand, when glucose supply is low, r is down-regulated (migratory phase; down-regulated m; up-regulated a) and [mass s ] exceeds [mass] influencing the cells to enter into resting phase g [ ] . let us assume that glucose (g) and drug (d) can be regulated through intravenous infusions and can be periodically administered. for illustrative purposes, consider h infusions every h with maximum dosage of unit, and refer this as regular infusion. we then have the intracellular dynamics under regular infusion can be seen in fig . since glucose and drug levels oscillate, it follows that m and r also periodically fluctuates around the threshold (th m � . , th r � . ) as can be seen in fig a. note that when m and r crosses the threshold value from above, respectively, peak in pseudo-mass is generated. it can thus be inferred that these peaks indicate cell migration since m and r levels are below their respective threshold values. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy as shown in fig b, a trajectory of core control concentrations in mtor-mir- -ampk space switches between proliferation and migration region. a closer look at the intracellular cell cycle proteins, mass, and mass s dynamics are illustrated in fig c. note that regular infusion significantly perturb the cell cycle dynamics stimulating several cell divisions (see mass profiles) and cell migration (see mass s ). indeed, fluctuating glucose levels in the microenvironment leads to the dichotomy of grow and go dynamics of glioblastoma cells yielding bigger tumor mass as reported in [ ] , even in the presence of (fluctuating) drug concentrations to keep the cells in proliferation phase. in the current investigation, we aim to regulate the amount of glucose and drug infusions to up-regulate mir- and mtor above its threshold values inducing cell proliferation strategies in regulating glioblastoma signaling pathways and anti-invasion therapy avoiding migration to neighboring tissues. the modeling approach utilizes optimal control theory to identify infusion administration protocols. let u g (t) and u d (t) be the controls of the system representing dose rates of glucose and drug intravenous administrations, respectively. two different administration protocols are examined, namely, ( ) concomitant, and ( ) alternating glucose and drug infusions, with the following objective functionals respectively. here, m(t) and r(t) denote the level of mir- and mtor concentrations, respectively. parameters b and b are weight factors measuring the relative cost based on maximizing mir- m(t) and mtor r(t), and administering glucose and drug intravenous infusions over a specified time interval, respectively. the control costs are modeled by the linear combination of quadratic terms u g ðtÞ and u d ðtÞ. our objective is to find optimal infusion regimen for glucose and drug administrations, denoted by u � g ðtÞ and u � d ðtÞ, such that the objective functionals are satisfied, that is, where the bounds for the controls represent the limits on dose rates for glucose and drug administrations. assuming that mir- , ampk, and mtor responses are regulated by glucose levels and influenced by drug levels, our control strategies deal with finding optimal control regimens for both glucose and drug intravenous infusions. we also note that the existence of optimal controls is guaranteed by standard results in control theory [ ] . in this maximization problem, the necessary convexity of the integrand in the objective functional holds. therefore, we can proceed with applying pontryagin's maximum principle [ ] . an iterative method is used for solving the optimality system which is a two-point boundary value problem having initial conditions for the state variables and terminal conditions for the adjoints. numerical simulations are obtained using a fourth-order iterative runge-kutta method. given the initial conditions and guess for the controls, state equations are solved using the forward scheme while the corresponding adjoint equations are solved using the backward scheme with the transversality conditions. the controls are updated by using a convex combination of the previous controls and the value from the characterizations. this is commonly referred as the forward-backward sweep method (fbsm) which is shown to be convergent [ ] . further details on the optimal control under study can be found in the s appendix. ( )) was performed in order to determine which parameters have the most/least influence on the reference output (main variables). all the core control parameters were considered to assess their corresponding influence on the mir- , ampk, and mtor activity. it was inferred that mir- activity will be enhanced by increasing glucose signal (g) and autocatalytic production rates (ℓ , ℓ ) of mir- . these parameters were negatively correlated with ampk activity due to the mutual antagonistic mechanism between mir- and ampk complex. in addition, ampk activity will be up-regulated by an increase in signaling source s and autocatalytic rates (ℓ , ℓ ) of the ampk complex in the model. it has been also noted that increasing s and the inhibition strength of mtor by ampk (γ) down-regulates mtor level. in a similar fashion, s is strongly positively correlated with mtor levels but little correlated with g, ℓ , ℓ , ℓ , ℓ , α, β, � , � at time . in this work sensitivity analysis is carried out to determine which model parameters have consequential effect in achieving or inhibiting bistability in the (g, r)−curve. as in [ ], a method from [ ] is adapted where a range for each parameter is selected and divided into n intervals of uniform length. for each parameter of interest, a partial rank the prcc values range between - and with the sign determining whether a change in the parameter value will promote (+) or suppress (−) bistability. the following algorithm is performed: . assign a probability distribution d½m j;min ; m j;max � to each parameter μ j and let n be the number of samples to be selected. divide the interval [μ j,min , μ j,max ] into n equiprobable subintervals, and draw an independent sample from each subinterval. . assemble the lhs matrix l, wherein each row of l represents a unique combination of parameters sampled without replacement. . for each row of the lhs matrix l, solve for mtor r in terms of glucose g and check for bistability window w b . if bistability exists assign to output variable w b in matrix y, else assign − . . rank-transform the matrices l and y to obtain l r and y r . by rank-transform, we mean to replace the value by its rank when the data are sorted from lowest to highest, e.g. the smallest value is assigned a rank . tied values are assigned an average rank. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy . fix a parameter μ j , which is encoded in the j th column in the matrix l r . form the following linear regression models using the data matrices l r and y r for μ j and y, respectively: compute ðm j Àm j Þ and ðy ÀŷÞ, the residuals in the input parameter and the output after removing the linear effects of the other input parameters. . obtain the prcc of μ j using prcc m j ;y ¼ covðm j Àm j ; y ÀŷÞ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi varðm j Àm j Þvarðy ÀŷÞ . repeat steps and for the remaining parameters. it illustrates that a change in the mir- autocatalytic production rate (ℓ ) or inhibition strength of ampk complex by mir- (β) enhances bistability. on the contrary, the hill-type coefficient ℓ in the regulation of ampk is responsible for losing bistability. it should be noted that due to the model's structure, ℓ up-regulates mir- which in turn increases mtor and suppress ampk activity. the parameter ℓ promotes ampk complex and down-regulates mir- and mtor. however, other parameters ℓ , α, ℓ , s , ℓ , ℓ , γ, s are little sensitive in emergence or destruction of the bistability. the bistability of mir- -ampk-mtor core control system depends on the geometric structure of its nullclines. in particular, bistability arises when m-and a-nullclines (i.e., dm dt ¼ and da dt ¼ ) intersect at three distinct points, producing one unstable and two stable steady states. the nullclines intersect three times due to their sigmoidicity influenced by catalytic rates ℓ and ℓ . these rates must be proportionate for a given glucose g level. otherwise, the nullclines will intersect only once. this bistability condition has been shown for a genetic toggle switch in e. coli [ ] . fig a- c depict the m-and a-nullclines under different g levels with various ℓ and ℓ values. the bifurcation curves for ℓ , ℓ and region of bistability for specific g levels are depicted in fig d. under given circumstances, ℓ and ℓ showed significant sensitivities in the bistability of the system. on the contrary, both ℓ and ℓ affect the r-nullcline only. it is illustrated in fig e that for several ℓ and ℓ values, r-and a-nullclines intersect at only one point, leading to a single steady state. in effect, m-and a-nullclines will also intersect once, producing monostability. hence, ℓ and ℓ show insignificant consequence in achieving bistability (see fig ) . in the following numerical experiments, it is assumed that initially glioma cells are in growth phase (a probable occurrence of post primary tumour surgery) with m > th m , a < th a , and r > th r . it is also considered that glucose and drug can be administered exogenously as intravenous infusions. further, the weight parameters b = and b = are used as default values unless specified. in this strategy, glucose and drug infusions are administered simultaneously, in particular, both controls u g (t) and u d (t) are infused at the same. thus, this is referred to as concomitant control. here, it is assumed that the drug in consideration which blocks the inhibitory pathway of mtor by ampk complex had negligible side effects and had inconsequential chemical reactions with glucose. in order to determine an efficient strategy of concomitant infusion protocol, glucose and drug are administered concurrently at initial time t = for hours using the numerical scheme fbsm. infusion spontaneously increases glucose and drug concentrations as depicted in fig (a) and (b) . consequently, mir- and mtor levels are up-regulated while ampk complex is down-regulated as shown in fig c. a closer look at the control curves shows that both u g (t) and u d (t) decrease from < t i < suggesting that both glucose and drug dose rates should be decreased from time t i . this leads to the decrease of glucose and drug concentrations due to consumption and decay. accordingly, mir- and mtor levels decrease while ampk complex increases. before mir- crosses the threshold value th m , fbsm is again applied for the next hours. this suggests a time for the next concomitant administrations in which mir- profiles are monitored subsequently. the procedures in tracking mir- profiles and applying fbsm for hours are repeated over a specified time duration. thus, the number of concomitant administrations are determined. it is important to note that keeping mir- level above its threshold value consequently confine ampk and mtor levels, below and above its corresponding threshold values, respectively (see fig c) . suppose that concurrent glucose and drug administrations is not plausible due to unwanted chemical reactions. we propose another control strategy that administers glucose u g (t) and drug u d (t) infusions alternately. at initial time t = , glucose infusion is obtained by solving the optimal control problem using the numerical scheme fbsm for hours. next, drug infusion is attained for the next hours in a similar manner. hence, the controls u g (t) and u d (t) are applied one after the other. subsequently, mir- levels are then tracked and before it crosses the threshold value, glucose infusion u g (t) and drug infusion u d (t) are again administered alternately in a similar fashion above, over the specified duration of administration. thus, the time for the next alternating infusion is determined by tracking mir- levels. this strategy is referred simply as alternating control. this infusion protocol is shown in fig (a) and (b). note that glucose infusion increases mir- levels and drug infusion activates mtor activities keeping ampk down-regulated as depicted in fig c. the intracellular dynamics under alternating control is illustrated in fig . as shown in fig a, both concomitant and alternating control strategies are able to sustain elevated mir- and mtor levels above their threshold values and ampk levels below its threshold value. it was also shown that mtor-mir- -ampk trajectory is restrained in the proliferation region prohibiting cell migration. further, number of cell divisions is reduced with slightly higher mass concentration before division compared to regular infusions but lower compared to constant (intermediate) high glucose concentrations. in this section, we compare the cost efficiency of the proposed strategies in terms of frequency of administration, dose per infusion, total glucose and drug amount, relative cost per infusion, and total cost incurred in the administrations. for the following results, the time for simulation duration is considered to be hours ( days). recall that parameters b and b are the weight factors associated in our objective functional which represent the measure of costs involved in the administration of glucose u g (t) strategies in regulating glioblastoma signaling pathways and anti-invasion therapy and drug u d (t) infusions, respectively, which also includes dosage, type, brand, medical fee for administration, etc. fig a shows that as the cost of glucose administration becomes expensive (increasing b values) with fixed drug administration cost (b = . ), frequency of concomitant and alternating infusion increases. however, it should be observed that as frequency of administration increases, the optimal glucose dose per infusion decreases with drug dose per infusion increases, see fig b. increasing drug dosage compensates the decreasing glucose dosage in order to keep mtor activities up-regulated leading to cell proliferation. on the contrary, if drug administration cost increases (increasing b values) with fixed glucose administration cost (b = . ), frequency of administration remains almost constant. this is depicted in fig c. the glucose dose per infusion is almost the same but the drug dose per infusion decreases with increasing b as illustrated in fig d. further, note that frequency of concomitant infusion control is always lower than that of alternating infusion control. in addition, drug (glucose) dose per infusion of concomitant control is always more (slightly less) than that of alternating control. for increasing glucose administration cost (increasing b ) with fixed drug administration cost (b = . ), total amount of glucose used for both control strategies generally decrease (except for high b > . values), as depicted in fig a, while total amount of drug increases, see fig b. on the other hand, when drug administration becomes expensive (increasing b strategies in regulating glioblastoma signaling pathways and anti-invasion therapy values) with fixed glucose administration cost (b = . ), the total amount of glucose dosage is almost constant (just slightly increasing for concomitant control) as shown in fig c, but the total drug amount is decreasing as can be seen in fig d. as illustrated in fig , concomitant control use less total amount of glucose than that of alternating control. contrarily, concomitant administration consumes more total drug amount as compared to that of alternating control (except possibly for high glucose administration cost, b > . , see fig b) . figs and reflect the relative cost per infusion and total administration cost of glucose and drug infusions incurred under concomitant and alternating controls. with fixed drug administration cost (b = . ), relative glucose cost per infusion and total administration cost increases as b increases. note that alternating control cost for glucose is always higher than concomitant infusions, see fig (a) and (b) . the relative and total administration cost for concomitant infusion slightly increase as compared to alternating control as b increases. again, alternating control incur more cost for higher glucose administration cost as illustrated in fig (c) and (d) . on the other hand, when b = . and drug administration cost (b ) increases, the relative glucose cost per infusion and total administration cost is almost constant. this can be seen in fig (a) and (b) . again, relative total cost for alternating control is higher than that of concomitant control. further, as b increases, relative drug cost per infusion and total administration cost decreases (fig (c) and (d) ), since total drug dosage also decreases as shown in fig d. in this case, it can be seen that drug administration cost for alternating control is generally lower than that of concomitant control. observe that in fig , both concomitant (blue) and alternating (orange) control trajectories in glucose-mtor-drug space are restricted in a smaller region avoiding aggressive invasion, rapid proliferation, and unwanted drug complications. both strategies achieved the goal of keeping mtor (mir- ) up-regulated inducing cell proliferation and thus avoiding aggressive cell migration. it is important to note that under these proposed optimal control infusions, glucose and drug levels are regulated to prevent excessive cell division and tumor growth. as a consequence, these strategies suggest safer infusion administration preventing hyperglycemia for diabetic patients and risk of drug complications. table provides the average frequency, dosage and relative cost of concomitant and alternating control strategies where b = b = . and simulation time is h ( d). the periodic switching behavior of glioblastoma cells between proliferation and invasion phases is highly influenced by fluctuating glucose levels [ , ] . in response to high glucose supply, mir- and mtor are up-regulated and ampk complex is down-regulated inducing cell growth. on the contrary, low glucose level up-regulates ampk complex, down-regulating mir- and mtor, promoting cell migration [ ] . the mutual antagonistic mechanism strategies in regulating glioblastoma signaling pathways and anti-invasion therapy between mir- (mtor) and ampk complex and the cell's strategic metabolic adaptation support the survival of cancer cells even in a nutrient-deprived microenvironment [ , ] . in addition to rapid proliferation of glioblastoma cells, aggressive invasion to the surrounding tissue is a major cause of treatment failure. despite advances in medical imaging technology such as mri and pet, glioblastoma cells can spread beyond detection leading to tumor recurrence within to cm of the resection cavity even after surgical removal of a malignant glioblastoma [ ] . these glioma cells are capable to deform cell membrane and nucleus for cell infiltration through a narrow gap between normal glial cells in brain tissue by upregulation of myosin ii along with actin bundles [ , ] . while exact migratory patterns are still poorly understood, these invasive glioma cells prefer white matter and blood vessels [ , ] with a wide range of speeds in the range of - μm/h [ ] [ ] [ ] [ ] showing sometimes saltatory patterns in the migration direction in brain [ ] . prediction of tumour invasion directions in nearby tissue may help define exact boundaries of focal treatments (surgery or radiosurgery), preventing future growth and recurrence [ ] . for example, medical doctors could determine specific locations for radiation target volumes, not just using a rough estimate of - cm in all directions as a guidance as commonly done today [ ] . assuming that migratory cells are localized near the surgery site [ ] , one possible approach is to keep the cells in its proliferative phase preventing them from invading brain tissue in a combination with transport of therapeutic drugs near blood vessels [ ] . as a result, the tumor strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mass will be visible for a succeeding surgery while killing proliferative tumor cells at the blood sites. in this study, we considered the intracellular dynamics of the mir- -ampk-mtor-cell cycle signaling pathway model developed recently by kim et al. [ ] . incorporated in the model is a drug component which blocks the inhibitory pathway of mtor by ampk complex. this drug targets up-regulation of mtor activities enhancing cell proliferation. the focus of the current work is to regulate up-stream signaling pathway via glucose infusion activating strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mir- , and control the down-stream pathway to cell cycle via drug infusion enhancing mtor activities. optimal control problem is formulated with the goal of keeping high levels of mir- and mtor to induce cell growth and reduce invasion to the surrounding tissues. in the framework of optimal control theory, two administration strategies are explored to achieve the goal with minimal cost incurred in glucose and drug administrations. the control strategies investigated in this study are ( ) concomitant infusion control and, and ( ) alternating glucose and drug infusion control. both strategies are able to switch on the proliferative mode of glioblastoma cells and turn off its migratory mode. cell cycle is regulated with fewer mass divisions restricting rapid growth. numerical results show that concomitant control had fewer infusions, lesser glucose dosage and cheaper administration cost. however, when glucose and drug poses unwanted chemical reactions during concurrent administration, alternating control would be beneficial with lower drug amount usage. the mathematical models of the mir- -ampk-mtor core control [ , [ ] [ ] [ ] ] are based on a 'go-or-grow' hypothesis and supporting experiments in gbm cells [ , , ] . however, the range of bistable behavior indicates a 'go-and-grow' program which has been proposed for other cancers too [ ] . in the glioma cases, 'go-or-grow' hypothesis has been long suggested [ , , [ ] [ ] [ ] [ ] in addition to mir- -induced 'go-or-grow' mechanism [ , , ] . glioma consists of a bulky, proliferative core in the center and highly invasive individual cells in the outer rim [ , , , ] and sequential transition between proliferative and invasive phenotypes characterizes tumor progression and may lead to faster growth [ ] . at least at the microscopic level, these migration/proliferation phenotypes appear to be mutually exclusive characteristics at different time frames [ ] , as suggested by in vivo imaging data of glioma cells migrating in a saltatory fashion [ ] . glioma cells was shown to pause for a short period of time (�an hour) for cell division before the daughter cells begin to move again [ ] . gal et al. [ ] further experimentally observed that this phenotypic switch can happen under different extracellular environment: (i) proliferative type in soft agar via activation of myc signaling pathways in response to hepatocyte growth factor (hgf); and (ii) infiltrative type in matrigel through ras signaling pathways in response to the same hgf. recently, dhruv et al. [ ] also affirmed the role of activation of key molecules in dichotomy between proliferation and invasion: c-myc and nfκb in the proliferative core and radially dispersed, invasive region of gbm tumors, respectively. glial cells can also interact with gbm tumor cells for phenotypic switch of gbm cells between cell migration and active proliferation [ ] . although these experiment and mathematical modeling support the 'go-or-grow' hypothesis, it is not certain if alternative mechanism such as 'go-andgrow' occurs in such a heterogenous tme in real patients [ ] . mansury et al. [ ] for instance illustrated that individual glioma cell in a mixed group of proliferative and invasive phenotypes can depend on genotype of counter part and tumor microenvironment. in a similar conceptual studies on epithelial-to-mesenchymal transition (emt) and mesenchymalto-epithelial transition (met) [ ] can give a hint on the complexities of this complex regulation of those two phenotypes and glioma in tme might not posses the simple 'go-or-grow' dogma. increasing number of evidences now suggest that the 'go-or-grow' model has similar molecular basis with emt [ ] . for instance, hgf and tgfβ are major regulators of emt, and these also provide strong stimuli of gbm invasion [ , ] and, upregulation of met and cd activities, as well as an activated nfκb signaling pathway, was reported in both mesenchymal gbm and metastatic cancer [ ] [ ] [ ] [ ] . all these experimental observations suggest that metastatic epithelial cancers and mesenchymal gbm drive common mechanisms that regulate the phenotypic transition between invasion and proliferation, suggesting the possibility of the 'go-and-grow' mechanism. the emt paradigm in gbm has not been extensively studied as relevant to the progression of the disease due to different origin and rare metastasis of glioma [ , ] . further studies and experimentation need to be done for better understanding of the fundamental principles. in this paper, we did not take into account key microenvironmental factors such as endogenous immune dynamics including nk cells [ ] and m /m macrophages [ ] , other major signaling networks [ , ] such as e f and myc [ , ] , angiogenesis [ , ] , biophysical interaction between tumor cells and blood vessels [ ] , ecm remodeling for therapy [ , [ ] [ ] [ ] , or growth factors [ , ] such as epidermal growth factors [ , , ] , fibroblast growth factors [ ] , transforming growth factor-β [ , ] , and csf- [ , , ] , that may play critical roles in proliferation, progress, aggressive invasion of gliomas and development of anticancer strategies [ ] . for example, endogenous nk cells may interfere oncolytic virus combination therapy in gbm while exogenous nk cells increase anti-tumor efficacy [ ] , illustrating the complex nature of tumor microenvironment. recently, mtor was considered to be a master regulator of cell growth and recognized as a good therapeutic target for therapies in glioblastoma [ ] . a recent study found that withaferin a and temozolomide can induce apoptosis and reduce drug resistance by g /m cell cycle arrest through intrinsic and extrinsic apoptotic signaling pathways [ ] . more detailed analysis and experiments on akt/mtor/pi k are necessary. interestingly, radiation was shown to indirectly promote the export of lactate into the extracellular space and inhibition of ampk/nfκb signaling pathways were involved in radiation-induced invasion of cancer cells [ ] . on the other hand, m microglia/macrophages induce matrix remodeling and glioma cell invasion [ , [ ] [ ] [ ] . since pi k signaling was shown to contribute to m -polarization of these tumor associated macrophages (tams) [ ] and pi k binding to csf r was shown to enhance spreading of macrophages, thus promoting glioma cell invasion [ ] . therefore, better understanding of these pi k-mtor-csf r signaling networks in macrophages would lead to development of blocking aggressive infiltration of cancer cells. signaling pathways of apoptosis and necroptosis are important parts of oncolytic virus (ovs) therapy [ , ] . tumor extracellular matrix (ecm) plays a significant role in regulation of glioma invasion in brain tissue as well as ov spread [ ] . for example, cspgs, one of major tumor ecm in brain can characterize the invasive and non-invasive gliomas in a complex tme where microglia and astrocytes mechanically interact with cspgs and tumor cells [ , [ ] [ ] [ ] . hybrid models [ , [ ] [ ] [ ] and its associated optimal control strategies can be adapted to take into account this important intracellular signaling as well as cell population dynamics and tissue dynamics. better understanding of various roles of these components in tumor microenvironment may provide better anti-invasion strategies of glioma cells. however, our mathematical model in this work is a first step toward further experimental/ clinical investigation and more optimal anti-invasion strategies of gbm by incorporating these microenvironmental components. we will address these issues in future work. supporting information s appendix. optimal control problem. formulation of the optimal control problem for concomitant and alternating glucose and drug infusion. 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bortezomib-induced unfolded protein response increases oncolytic hsv- replication resulting in synergistic antitumor effects bortezomib treatment sensitizes oncolytic hsv- treated tumors to nk cell immunotherapy choindroitinase abc i-mediated enhancement of oncolytic virus spread and anti-tumor efficacy: a mathematical model chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment contributions of chondroitin sulfate proteoglycans to neurodevelopment, injury, and cancer glycosaminoglycans and glioma invasion a hybrid model for tumor spheroid growth in vitro i: theoretical development and early results multiscale models of cell and tissue dynamics the role of the microenvironment in tumor growth and invasion this work is resulted from the konkuk university research support program (e.j.). this paper is also supported by the korea national research foundation (nrf) grant funded by the korean government (mest): nrf- r a b (e.j.) and the basic science research key: cord- - yas yqt authors: yoshikawa, minako jen title: dengue and chikungunya virus infection in southeast asia: active governmental intervention in republic of singapore date: - - journal: current topics of infectious diseases in japan and asia doi: . / - - - - _ sha: doc_id: cord_uid: yas yqt this paper discusses countermeasures of republic of singapore towards mosquito-borne infectious diseases, particularly, dengue and chikungunya virus infection to identify an essential factor in controlling emergence of infectious diseases. in spite of expanding areas affected by and upsurge of these diseases in the region, the tropical urban country is known to have sustained an effective vector control, which often resulted in moderate prevalence and/or quick control of domestic outbreaks. this research has adopted an inter-disciplinary review of previous studies combined with field studies: interviewing at the ministry of health, singapore and the national environment agency, singapore; visiting a laboratory and hospitals; and observing on-site vector mosquito surveillance operations conducted by the agency. the findings have pointed out the national vector surveillance and control system implemented by s, followed by improved countermeasures like vector and virus surveillance which have incorporated science and technology especially in the last two decades. the analysis produces an influential role of a government in promoting and supporting public health measures, which have been typically demonstrated through inter-ministry collaboration, public-private cooperation, and community involvement. in light of increasing transnational nature of emerging infectious diseases, singapore’s contribution in the region like sharing its knowledge of and experiences in dengue and chikungunya virus infection is illustrated. the resilient model of singapore’s vector control and governmental action warrants a further study to investigate transferability in other parts of the region. one can find in singapore a government that desires to protect health of the people while preserving economic and trade activities in the ages of globalization. singapore has maintained an impressive track record in responding to emerging and re-emerging infectious diseases (hereinafter expressed as emerging infectious diseases). for example, it was the year of when the country attracted waves of praise as a successful country in handling the outbreak of severe acute respiratory syndrome (sars). also, singapore's resilient vector control management for mosquito-borne infectious diseases is noteworthy. the "energetically sustained" dengue control program is acclaimed as "a model for the rest of the world" [ ] . although a considerable number of studies on its outbreak response, surveillance, prevention, and disease management have been conducted by the medical and science community, others often attribute singapore's outstanding infectious disease control to the small territory and economic affluence alone. medicine and science-oriented studies, too, tend to pay little attention to factors that are not scientifically proven. what seems to be lacking is an inter-disciplinary investigation of emerging infectious disease countermeasures. this paper attempts to elucidate an active role of the singapore government in facilitating and sustaining satisfactory response to emergence of infectious diseases. the government here refers to the governing body consisting of politicians and the public servants engaged in the service of the supreme authority. this paper limits the discussion to singapore's action against dengue fever (df), dengue hemorrhagic fever (dhf)/dengue shock syndrome (dss) (hereinafter abbreviated as dengue infection) and chikungunya viral infectious diseases. we will first review how the young nation, having become an independent state only in , came to establish its national vector surveillance and control system by 's. next, elaborating singapore's measures in the recent decades, we reveal the heavy emphasis placed on the scientific research in public health tools in the country, which has been remarkably evident since 's. finally, we will devote some space to the discussion of singapore's role and capacity in a regional effort to control dengue infection. situated km north of the equator, highly populated singapore is often referred as a tropical city-state. population density was , /km , heterogeneous singapore citizens and permanent residents represented % of . million total population (mid-year estimates, ) [ ] . besides frequent travels of residents, mobility of people in singapore is marked by transnational movements, especially considerable influx of foreign labor and overseas travel. in fact, residents recorded just . % of labor force participation [ ]. about . million tourist arrivals (excluding malaysians arriving by land) show as high as % come from asia, one-half of which are from asean member countries [ ] . being a regional hub of frequent movements of people as well as trades and economy, the congested urban country pays careful attention to mosquito-borne infectious diseases spreading in the region. with constant import potential of viraemic humans, its hot and humid tropical climate of singapore permits another threat: propagation of mosquitoes throughout the year including aedes aegypti and ae. albopictus that serve as vectors of dengue and chikungunya viruses. chikungunya virus (chikv) is a single-stranded rna virus (family togaviridae, genus alphavirus) and is known to be transmitted by the same vector species causing dengue infection. as of march , countries have reported domestic chikv infection and the list includes almost all southeast asian countries including singapore [ ] . section . summerises singapore's response to the first and subsequent domestic outbreaks in . the agent of dengue infection is also a small single-stranded rna virus (family flaviviridae, genus flavivirus). a dengue infection cycle starts when a female aedes mosquito bites an infected human, acquires competence to mediate, and then bites another person(s). there are four serotypes of dengue virus, den- , - , - , and - and infection with one serotype could provide a lifetime protection to the particular serotype. cautions are, therefore, required against the subsequent infection with other serotypes. the world health organization (who) has analyzed factors contributing to the global burden of dengue infection. examples are rapid population growth, migration between rural and urban locations, and transnational travel by infected persons and goods [ , ] . the international health authority often cites that the vector control is the sole preventative tool currently available and maintains that "[d]espite the availability of effective tools to control dengue, efforts to prevent and control dengue have been constrained due to lack of sustained political commitment, inadequate resources and lack of coordinated effort" [ ] . the present study has benefited from inter-disciplinary research of previous studies and fieldworks between september and august . research activities during the field trips to singapore included: document collection; interviews with ministry personnel from the ministry of health (moh), health promotion board under the moh, and the national environment agency (nea) under the ministry of the environment and water resources; visits to the nea's laboratory called environmental health institute (ehi) as well as public and private hospitals; and observing two onsite inspections of the nea. particularly insightful were the larval surveillance and adult trapping operations performed by the south east regional office of the agency. the operations covered private houses, public highrise residential buildings (known locally as hdb flats, figure ), and a construction site of a condominium ( figure ) in september and march [ , ] . in addition, the author attended the inaugural asia-pacific dengue program managers meeting convened by western pacific regional office of the who (wpro) and co-sponsored by the nea in may [ ] . in august , the author visited the regional office of the wpro in manila, the philippines to learn views and activities of the international health authority. result: singapore's resolute efforts to fight against dengue infection known as breakbone fever, df has been prevalent for centuries and dhf first occurred in the philippines in [ ] . the first dhf outbreak struck singapore in , one year after it attained a self-government status ahead of the country's birth as an independent state in . the vector control unit (vcu) was formed in initially under the moh and was later transferred to the ministry of the environment in [ ] . goh kt ( ) elaborated activities of the vcu: it gathered ecological and biological data such as vector distribution and density, location of breeding habitats, and seasonal fluctuations in vector population. chan kl ( ) pointed out that several entomological surveys of slum areas around that time suggested the evidence of huge ae. aegypti inhabitants, supporting a close correlation between dhf and poverty [ ] . evidently, this finding led the young government to address the environmental issue, including replacement of slum houses and cleaning of residential areas. to deal with a challenging task to transform human behavior, the singapore government decided to use law enforcement and health education: the destruction of disease bearing insects act was enacted and the "keep singapore clean and mosquito-free campaign" was designed to motivate the community to participate in the countrywide vector control program in [ ] . in addition to the adult mosquito surveillance began in , a weekly aedes larval routine survey in premises became operational in june . subsequently, the incident rate of dhf dropped; morbidity rate per , was . in [ ] and remained between and for the period from to [ ] . however, optism resulting from low level of incidence might have translated into , cases with deaths in [ ] . the vector surveillance became even more crucial to establish the knowledge of "the normal level" of vector density, which enabled the vcu to calculate a premise index (pi) or a percentage of premises positive for aedes breeding to estimate dengue virus transmission risks. wherever a pi exceeded %, the vcu fogged the area with insecticides to eradicate the adult mosquitoes. as priorities of breeding habitats kept shifting owing to the vector behavioral change, the breeding sites elimination task became difficult in a cat-and-mouse game. to address the issue, the repeated evaluation and feedback system was used in s and s, which became useful to review and amend strategies ( figure ). in addition, the regulatory requirement made dengue infection legally notifiable in under the infectious diseases act of while dhf had been already made administratively notifiable in [ , , ] .while the rest of the southeast asian region experienced epidemics in and , the vector control system contributed to low frequency of dhf in singapore [ ] . singapore benefited from the reduced vectors for a period of almost years after recording cases in . its vulnerabilities to importation of dengue infection, however, existed as long as the disease was endemic in the region [ , ] . by the s, the city-state saw the resurgence of dengue infection. in addition to the expansion and escalation of the diseases in the region, the previous diligent vector control possibly fueled the situation as the low prevalence of the disease generated the large susceptible human populations due to reduced herd immunity [ ] . there was, however, another contributing factor: by then, the labor intensive vector control had been changed to respond to human cases instead of a higher pi in response to a call for efficient usage of public resources [ ] . areas "at risk" may not be sufficiently discovered by case detection [ ] as ooi ee et al. ( ) has illustrated: this shift inevitably overlooked risks that were hidden by subclinical cases [ ] . seeing the dengue infection increase from , cases in to , in , and to , in , the government set up in an educational program and launched volunteer groups. active persuasion of the community began. a political leader's involvement was evident in the following quotation: the best way to protect yourself and your family from dengue fever is to make sure that the mosquitoes do not have a chance to multiply in your homes and neighborhood. unfortunately not enough residents make the special effort necessary to prevent aedes mosquitoes from breeding…residents should therefore come together as a community to get rid of the mosquitoes, and keep the estate free from dengue fever. the above speech was delivered by then deputy prime minister and current prime minister lee hsien loong at the launch of an educational program and volunteer groups in may, [ ] . from this passage, we can infer the political will to solve the problem by influencing the residents to become more responsible and cooperative towards governmental intervention. subsequently, the number of cases declined to , in , in , , in , and , in [ ] . singapore was able to transform human behavior towards aedes mosquito breeding [ ] . however, as transmission outside home premises like construction sites emerged [ ] , the number of cases jumped to , in and , in [ ] . the large outbreak in occurred concurrently with the region-wide epidemic [ ] . by then, singapore was even more congested, experiencing nearly doubled population density from , / km in to , / km in [ ] . replacement of dominant dengue serotypes over the years, "a contributory role" of the rising temperature [ ] as well as cumulative precipitation in years - [ ] had been also documented. laboratory confirmed cases amounted to , comprising , df and dhf; fatalities represented the third largest among all notified infectious diseases in singapore in . several initiatives deployed and countermeasures reinforced in response are detailed in section . . when the city-state achieved lowering the cases significantly in , the who referred singapore as "a good example of a country that has successfully controlled the mosquito population…" [ ] . reported cases of , ( , df and dhf) in represented a decline of % from [ ] and all cases were confirmed by laboratories with one or more tests like anti-dengue igm antibody, enzyme linked immunosorbent assay (elisa), and polymerase chain reactions (pcr) [ ] . the figure included , indigenous, imported, and cases suffered by foreigners of whom the majority is patients having acquired the diseases overseas and sought medical treatment in singapore [ ] . although the territory wide anxiety was heightened in singapore when the weekly reported cases indicated as high as during july - th, , the weekly number in and remained below both the epidemic threshold of weekly cases and the warning level of [ ] . for dengue infection, communicable disease surveillance branch at the moh handles surveillance of human cases while the nea is responsible for environmental surveillance. in response to an alarming upsurge, the moh formed an expert panel in september . local and international experts of the panel reached a conclusion that the regular importation of dengue viruses into singapore was likely to continue "because dengue is a re-emerging disease globally and is endemic in the surrounding regions" [ ] . furthermore, the inter-agency task force was formed around the same time to enhance the communication and coordination among various agencies with the aim to strengthen the countermeasures in the areas of investigation, audit, and infrastructure [ ] . environment heath department of the environmental public health division of the nea is in charge of vector surveillance, control, prevention, and research operations. as many as , residential and , nonresidential premises on average are inspected each month [ ] . devices known as ovitraps are deployed across the city-state; an ovitrap is a black plastic container designed to attract female mosquitoes to lay eggs and then trap the emerging adults within the containers. the weekly checks of the ovitraps enable the nea to detect the presence and types of mosquitoes early and proactively remove mosquito breeding habitats [ ] . the department has compiled examples of common and potential mosquito breeding places by types of premises and sites to draw further attention from residents and visitors [ ] . in situation room at the head office, the nea uses a geographical information system (gis) implemented in for the analysis of the distribution of both vectors and infection cases. in december , the agency reinforced its vector surveillance and control system; about officers were conducting routine ground surveillance across the island especially in dengue-prone areas (e.g. construction sites, compounds of landed housing properties, and schools) for the purpose of detection and removal of breeding sites [ ] . in , the nea carried out inspections of approximately . million premises in addition to over , ground surveys. furthermore, the nea led two intensive source reduction exercises in april-may and july-september [ ] . the singapore authorities make the emergence of infectious diseases in the territory known in a timely manner. as for dengue infection, locations of cases and the results of outbreak investigations are made transparent. the moh also periodically communicates: moh medalert reports outbreaks; moh weekly infectious disease bulletin reveals weekly data; epidemiological news bulletin analyzes quarterly trends and special topics; and communicable diseases surveillance provides annual overviews and comprehensive data. additionally, news and updates of the outbreaks are often issued as press releases. all these materials mentioned above are made available on-line. as for the accuracy of the number of reported cases, the following section will clarify. both residents and visitors often see posters and other warning materials throughout the country especially when dengue cases are on the rise (figure ) . thus, it is reasonable to say that singapore's information is kept widely open to both local and international community. while many southeast asian countries tend to report only the manifested cases based on clinical diagnoses in general, singapore is known to report dengue infection inclusively (df/dhf/dss). thus, the variance in reporting system makes it difficult to compare the reported dengue cases for evaluation among countries in the region. this problem may be partly attributable to underdeveloped costly technology in the region. as for singapore, there is enough evidence to identify dedication of the government to science and technology development, especially following national science and technology plan announced in september . the singapore government prioritized its policy to make singapore "the hub for life-science research in southeast asia" [ ] . the political commitment can be seen as embodied in the grand establishment in : a complex of seven buildings named biopolis houses the biomedical industry including the ehi, the nea's laboratory. singapore regards laboratory-based virus surveillance as an integral part of the dengue countermeasures. it is important since identification of the antigen and four serotypes could produce scientific data that have significant relevance to epidemiological investigation and disease surveillance. in other words, the findings could suggest or predict potential shift of dominant serotype, which would cause large outbreaks. the ehi adopted a new economical method in called the multiplex rt-pcr technique with high throughput capability for the screening of dengue viruses [ ] . the technology can also facilitate rapid and reliable diagnosis to administer proper clinical treatments [ ] even when patients present undifferentiating febrile illnesses. moreover, an ehi researcher revealed faster and greater horizontal and vertical flight ranges of the dengue vector mosquitoes by conducting "mark-release-recapture" studies at three different locations in the city-state [ ] , which supplied useful information for vector control operations. in , researchers in singapore published a diagnostic algo-rithm allowing early diagnosis or differentiation of dengue infection with an accuracy of almost % from other febrile diseases [ ] . it can be said that each achievement is a product of government-driven investments in science for health, which singapore has been active since s. one recent addition was the s$ million grant announced in october ; the government of singapore will make available over five years to researches on dengue infection. the research efforts are expected to focus on the development of evidence-based case management and disease prevention and pursue to fill in gaps between bedside and "bench research" [ , ] . the battle against dengue infection of the nea is constituted of not only the vector control and virus surveillance, but also law enforcement and community involvement. legal compliance of dengue control measures is facilitated through the environmental public health act and the control of vectors and pesticide act. the former consolidates and defines the law pertaining to environmental public health and the latter makes it illegal to breed mosquitoes through the section of prohibition on creating conditions favorable to vectors. examples were (as of february ): s$ fine for the st and s$ for the subsequent offences committed in residential premises; heavier penalties are applied to construction sites as s$ , for the st, s$ , for the nd, followed by court appearance and maximum s$ , and/or six months imprisonment. the agency can ultimately order the construction companies to stop the work. [ ] . community involvement, on the other hand, is encouraged through various social activities and a series of campaigns. in when the author visited singapore for almost two months, an educational community outreach promotion or the indoor " -minute mozzie wipe-out" initiative which had been implemented in was actively seen. the community outreach effort by the agency appeared to ensure that knowledge was translated into practice: for example, it reminded the households to check and remove stagnant water at homes like water in flower vases [ ] . the educational materials prepared by the nea included pamphlets, booklets, posters, and audio-visual items in all four official languages of english, chinese, tamil and malay to reach residents, domestic help workers, and so on. materials addressing construction sites and shipyards showed additional three foreign languages as singapore has abundant influx of foreign labor as has been pointed out earlier. in addition, the standard operating procedures (sops) reveal singapore's action plans for ps, i.e. three sectors of people, private, and public (gov-ernment). the people sector covers grassroots organizations, residential clubs, ngos, and npos while the private sector means private businesses, associations, etc. the public could include government ministries and agencies listed in the singapore government directory but the particular dengue sops make more direct references to the nea in terms of source reduction and ulv/outdoor fogging. the sops have seven steps of action plans depending on the number of cases: , , - , - , - , - , and > cases [ ] . for instance, procedures adhered when two cases of human infection are identified in a particular area: a team of two nea officers gets deployed for vector control tasks; a member of parliament from the particular constituency and a community leader are informed to take actions; the moh warns doctors in the affected area; dengue alert poster/banner at the premises are displayed; grassroots organizations are engaged to speak to residents; grassroots members assist with distribution of dengue prevention pamphlets and alert letters to residents; and so on [ ] . an excess of , volunteers from the ps joined the outdoor "carpet-combing" exercise from september to october , and more than , aedes mosquito breeding and , potential sites were removed [ , ] . about , volunteers sacrificed their weekends to distribute the " -minutes mozzie wipe-out" pamphlets to nearly , homes [ , ] , indicating the community involvement in the governmental initiative. thus, the ps mechanism of human resource mobilization in singapore has proven effective; the national dengue response of the singapore virtually involves the whole nation. the year brought singapore a new challenge as the first domestic outbreak of chikungunya fever arrived in january. impressively, singapore terminated the transmission within days. according to interviews conducted at the moh and the nea in march and may , a collaboration of the clinical network consisting of general practitioners and the reference laboratory, the ehi, had been implemented; an active surveillance on chikungunya fever started as early as in . in fact, blood samples tested negative for dengue virus by pcr were then tested for chikv. the careful and thorough preparation, partly in response to the outbreaks in other countries, may explain why singapore was able to react quickly in january (yoshikawa m.j. et al., manuscript in press). shortly after the first domestic case of chikungunya fever was identified owing to the collaboration, the authorities collected a total of , blood samples within meter radius in the area where the index case worked/resided and discov-ered additional cases [ , ] . as for the vector control, the nea destroyed over , mosquito breeding sites [ ] . however, subsequent domestic outbreaks started in june . interviews at the nea in march and august revealed that the previous control measures were not sufficient to address the new situation. as a phylogenetic analysis of chikv envelope (e ) gene by the ehi revealed, l.c. ng et al. ( ) confirmed that subsequent domestic outbreaks were much more complicated with importation of multiple chikv strains [ ] . furthermore, vector surveillance and control operations proved the new vector competence acquired by additional vector, ae. albopictus [ ] . the first domestic outbreak was mediated by ae. aegypti mosquitoes [ ] . whereas ae. aegypti tend to stay in and around residential premises and buildings, ae. albopictus breed in outdoor environments. thus, lowering the vector population quickly was a real challenge. indeed, the subsequent domestic outbreaks were located in the rural and sub-urban areas of the city-state [ ] . the revised vector control strategy incorporated these scientific findings; the agency increasingly utilized outdoor thermal fogging to kill adult mosquitoes, expanded target area to remove breeding sources, and intensified operations at areas with vegetation. clearly, the city-state benefited from its past and present dengue infection control experiences; the similar governmental intervention was carried out through the inter-ministry collaboration and public-private cooperation during the course of chikungunya fever domestic outbreaks ( global warming and the expansion of affected areas of mosquito-borne infection do not seem to be reversing anytime soon; the northern limit of dengue infection appears moving upward. ae. albopictus in japan raises concern. although its reported cases of dengue and chikungunya virus infection have been all imported for now, df epidemics broke out in the western japan between and [ , , , , ] . approximately % of imported cases during - period were traced to india, indonesia, the philippines, thailand, and malaysia [ ] . as of december , , imported cases of chikungunya fever were recorded in japan arriving from (the patient numbers in parenthesis): sri lanka ( ), india ( ) indonesia ( ), malaysia ( ), and thailand ( ) [ ] . as reiter ( ) claimed a decade ago that singapore was "situated in the world's major region of dengue transmission" [ ] , the city-state is at constant risk, given the significant number of tourist arrivals from the regional countries. in fact, situation in the asean countries deteriorated further in this century [ , ] . with the global expansion of the infected areas, the asia-pacific dengue partnership was set up in march under the wpro and the south-east asia regional office of the who (searo). the who has estimated that . billion populations residing in these two regions are at risk, bearing about % of the global burden of the disease [ , ] . although lower case fatality rates owing to better case management is recently observed in several member states, the disease is spreading not only to more countries but also from urban to rural areas [ ] . dengue infection, therefore, is unlikely to remain just an "urban problem". the wpro and the nea co-hosted the inaugural asia-pacific dengue program managers meeting in singapore in may . the event was attended by member states from the wpro and five from the searo in addition to temporary advisors, partner agencies, and observers [ , ] . the bi-regional initiative can be beneficial for the asean countries to work vis-à-vis the public health threat in the region as they belong to the two separate who regions. during the meeting, singapore presented informative analysis of the historical and current situation of dengue infection and integrated approach on vector control and virus surveillance. several participants praised its unique and outstanding inter-agency dengue task force as having enabled good resource mobilization [ ] . the first asia-pacific dengue workshop was also organized in singapore by the who and co-hosted by the ministry of foreign affairs, singapore and the nea in march . sharing field surveillance technique and laboratory research findings in the week-long capacity building workshop, singapore joined the who in offering expertise on mosquito-borne infectious disease control. in addition, the city-state is said to have provided assistance to cover training and material costs, medical insurance for the participants during the workshop, and accommodation for the entire duration of the workshop [ ] . we recall that community-based approach and program have been emphasized in various global issues of concern in the recent decades, including public health. it is indeed difficult to control infectious diseases without community involvement. however, in light of inherent transnational nature of emerging infectious diseases today, public health professionals are expected to apply effective control measures without seriously disrupting movements of people and goods, coordinate effort regionally and/or globally, and make the use of limited resources. it is necessary to keep in mind that curving mosquito-borne epidemics like dengue infection and chikungunya fever require capacities such as laboratory-based surveillance and territory-wide vector control program as well as regional collaboration. thus, the community-oriented approach is unlikely to accomplish the mission in the absence of respective government involvement. in this respect, as we have examined, singapore has the committed policymakers and the dedicated public servants who strive to sustain and improve infectious disease countermeasures. while there are occasional criticisms towards singapore's strict law enforcement as well as concerns for the dominant party rule, this paper has attempted to deduct an important role of a government in appropriate and successful intervention in emerging infectious diseases of global concern. from what has been seen in singapore's response to dengue and chikungunya domestic outbreaks, we can conclude that the strong governmental intervention of the city-state often manages to achieve support from the community. on the "neglected tropical disease" battlefield of dengue infection, it is inspiring to see the small tropical citystate 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- dengue epidemics of japanese main islands expansion of the habitation area of aedes albopictus, a dengue fever vector mosquito dengue fever dengue hemorrhagic fever and new findings viral titers in the sera of dengue patients among travelers at the quarantine station of kansai international airport national institute of infectious disease and tuberculosis and infectious diseases control division, ministry of health, labour and welfare, japan. imported dengue and dengue hemorrhagic fever in japan, as of world health organization regional office for the western pacific dengue: guidelines for diagnosis, treatment, prevention and control world health organization regional office for the western pacific this research was supported in-part by grant-in aid for scientific research (kakenhi ) by the japan society for the promotion of science. the author is especially indebted to all the individuals in singapore and at the who for making the research materials available and sparing their precious time for me to interview. key: cord- -w sh f h authors: xue, lan; zeng, guang title: china’s institutional mechanisms for influenza a (h n ) prevention and control date: - - journal: a comprehensive evaluation on emergency response in china doi: . / - - - - _ sha: doc_id: cord_uid: w sh f h innovation in institutional mechanisms is a fundamental issue in effectively dealing with public health emergencies. in the wake of the sars epidemic, china initially established a public health emergency management system and an emergency organization and management network, placing emphasis on “government leading, unified command, local management, responsibility on all levels, management by classifications, and inter-departmental coordination,” which strengthened the existing health emergency preparation system. and insufficient preparedness. china's fight against sars, as it were, not only posed a great challenge to the nation's socialist modernization, but at the same time offered an important opportunity for the country to improve emergency management, especially in the field of public health. in the wake of the sars epidemic, the chinese government began pushing for a national emergency management system in a systematic, planned and gradual manner, and made remarkable progress in emergency management structured on the "one plan three systems (contingency plans, institutions, mechanisms and legislation)." in regards to contingency planning, the country formed a system consisting of contingency plans at central, local, departmental, and enterprise levels as well as plans for major events, and this system played an important role in dealing with public emergencies. in regards to institution building, a national emergency management system consisting of general emergency management offices as well as of special emergency management bodies were established. the emergency management office of the state council and emergency management bodies of provincial (regional and municipal) governments were set up in succession, in addition to emergency management systems in specific fields such as health. in comparison with the pre-sars environment where "departments played a dominant role, and coordination was inadequate," these new institutions showed the permanent, comprehensive, and specialized nature of emergency management, and laid an organizational foundation for the future. in addition, society as a whole began getting involved with the emergency management process, including: further strengthening military emergency system construction and local assistance ; giving full rein to experts in in march , the third session of the th national people's congress (npc) reviewed and adopted the report on the work of the government, which stated that "… we have formulated the national contingency plan for public health emergencies, as well as special-purpose and department-specific contingency plans concerning natural disasters, accidents, public health, social security, etc.; provinces (regions and cities) also have completed their own work on the development of overall contingency plans. breakthroughs have been made in building a government by the rule of law and in fully fulfilling government functions." see: report on the work of the government, delivered by wen jiabao at the third session of the th national people's congress on march - , . emergency management ; and developing and implementing local emergency management plans that targeted "communities, rural areas, enterprises and schools." looking at mechanism construction, progress was also made in research on a science-based emergency management system, and an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation"-which enabled the interconnection of early warning, mass mobilization, quick response, and emergency handling-was gradually established to effectively mitigated public health emergencies. in regards to legislation, the emergency response law was took effect on november st, . health emergency system long term. efforts would be made also to strengthen the disease control and prevention system, increase public health emergency management capabilities, boost rural health development, improve healthcare for the rural population, strengthen environmental health system, and implement national health campaigns. in february , the moh outline the following overall goals for public health emergency work in the th five-year plan: establish and improve health emergency management legislation and the health emergency contingency planning system; build an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation" with "predominantly local management, hierarchical responsibility, and comprehensive coordination;" bolster health emergency management recruitment; improve the public health emergency monitoring and warning system; strengthen capacity for quick and effective response to health emergencies; and shape an environment of health emergency management characterized by inter-departmental coordination, collaboration, and social participation under the leadership of central and local governments. during and after the sars epidemic, china continued to establish and improve public health emergency legislation, regulations and contingency plans, and initially formed a national system for public health emergencies contingency planning (see fig. the moh established a public health emergency coordination mechanism with thirty one central and national departments to deal with inter-departmental collaboration, which effectively strengthened communication and coordination between departments dealing with public health emergencies. the national government and the special administrative regions of hong kong and macau entered into a three-party emergency response collaboration agreement and decided upon implementation regulations, and established a linkage mechanism for information communication and health emergency response. additionally, the moh established a joint prevention and control mechanism with the ministry of agriculture (moa) to protect against highly pathogenic zoonotic viruses such as avian influenza and streptococcus suis; the moh established a coordination mechanism for joint prevention and control of public health emergencies at ports with the general administration of quality supervision, inspection and quarantine (aqsiq). in collaboration with the ministry of railway (mor), the ministry of transport (mot), and the aqsiq, the moh issued notices on the prevention and control of the importation of infectious diseases from abroad; and together with the moe, issued a document requiring schools to appoint part-time or full-time teachers to identify and report infectious diseases or other health emergencies at the school. this time marked the initial formation of a working inter-departmental mechanism positioned to combat health emergencies through "paying equal attention to both prevention and response, and instilling continued collaboration for any event." on april th, health minister chen zhu convened a meeting of the moh leading group and expert panel for influenza pandemic prevention and control, at which the attendees analyzed swine influenza situations in the united states and mexico, predicted epidemic trends, and deliberated on domestic strategies and measures to cope with a swine flu pandemic. health minister chen also held an inter-departmental meeting with the moa, the aqsiq, and other ministries to analyze epidemic trends and discuss response strategies and measures. immediately after the meeting was over, the moh reported in writing that very night to the state council on the progress of epidemic prevention and control work. on april th, following the emergency meeting held in geneva, the who elevated the pandemic alert level from phase to phase , stating that the "swine flu" was widespread and was being transmitted by humans in different ways. general secretary hu jintao issued instructions to place prevention and control against this virus as the nation's top priority. on the same day, vice premier li keqiang convened the state council meeting regarding the human-swine influenza prevention working mechanism, resulting in the decision to establish a multi-departmental working mechanism for joint prevention and control of the human-swine influenza. as required by the state council meeting, the moh called together the publicity department of the communist party of china (ccppd), the ministry of foreign affairs (mfa), the ndrc, the miit, the mof, the ministry of transport (mot), the moa, the moc, the aqsiq, the china national tourism administration (cnta), the civil aviation administration of china (caac) among other departments on that very night for a meeting to deliberate on influenza a (h n ) prevention and control; the meeting established the multi-departmental working mechanism for joint prevention and control of human-swine influenza and the notice on strengthening human-swine influenza prevention and control was drafted and published on the night of april th after state council review. the moh issued the notice of the moh general office on strengthening preparedness for and response to human swine influenza. on april th, premier wen jiabao convened a state council executive meeting which deliberated on how to strengthen national response to human-swine influenza; at the meeting they defined the overall prevention and control principles and strategies of "taking threats to public health seriously, responding actively, and coping with the epidemic in a scientific manner according to law through joint prevention and control efforts." on april th, the who raised its pandemic alert level from phase to phase . on april th, at a press conference held at the state council information office, the moh declared the establishment of a multi-departmental working mechanism for joint prevention and control against the human-swine influenza, which would be spearheaded by the moh. under this mechanism, departments and institutions (which later increased to ) constituted work groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert committee, forming a " + " pattern for joint prevention and control efforts. on the afternoon of may st, the joint prevention and control mechanism held its second joint conference, renaming the human-swine influenza which was occurring in mexico and the united states to "influenza a (h n )." the former wording of "multi-departmental work mechanism for joint prevention and control of human-swine influenza" was changed to the "joint national influenza a (h n ) prevention and control mechanism" and roles and responsibilities were outlined for the mechanism, work groups, and the expert committee. at the same time, a meeting system for all members and liaisons was established. problems in principle which a work group encountered would be solved by the work group itself, and those which the work group struggled with would be settled through coordination under the joint prevention and control mechanism-general affairs would be solved by regular liaison meetings, and major issues decided by plenary meetings. health minister chen zhu and moh party group secretary and vice health minister zhang mao chaired the joint national influenza a (h n ) prevention and control mechanism, and the mechanism was comprised of eight working groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert advisory committee. the heads of the work groups, the leaders of the health department of the people's liberation army general logistics department (gld), the logistics department of the chinese people's armed police force (papf), and the chairman of the expert advisory committee, served as members of the joint prevention and control mechanism. the work groups, the gld health department and the papf logistics department each designated one or two departmental-level officials as liaisons for routine communication purposes. the main duties of the joint national influenza a (h n ) prevention and control mechanism included: meet regularly to evaluate epidemic trends and determine prevention and control strategies; formulate prevention and control policies, response plans and major measures; coordinate and provide guidance on the implementation by all related departments and in various regions of prevention and control measures; and organize supervision and inspection activities concerning the implementation of prevention and control measures. see table . for the main responsibilities of the work groups and the expert advisory committee. thirty three meetings were convened under the joint national prevention and control mechanism, in which regulations were formulated, signed, and issued for local implementation. implementation issues would be reported in real time to related state council departments for instructions. the establishment of the joint national prevention and control mechanism played a crucial role in the scientific and orderly response to influenza a (h n ), in that ( ) the mechanism raised the priority level for influenza a (h n ) prevention and control for related departments and local governments, ( ) clarified and divided responsibilities, ( ) addressed investment issues, and ( ) enhanced interdepartmental cooperation. a consultation system and a liaison meeting system were established under the joint national prevention and control mechanism, which were designed to ensure effective implementation of prevention and control measures. specific issues in prevention and control work would be resolved through consultation at liaisons meetings, and major issues decided by plenary meetings. each working group established a fixed meeting system where they could provide timely progress comprehensively coordinate routine affairs among departments of the joint prevention and control mechanism; organize regular meetings of the joint prevention and control mechanism and oversee the handling of top agendas; collect, sort out, and report to higher-ups about progress in prevention and control efforts; prepare progress reports on the joint prevention and control mechanism; publish information on epidemic situations and response efforts; ensure the consistency in style of writing for documents intended for outside use; and shoulder other assignments from leaders report in a timely fashion on influenza a (h n ) situations and on progress made in major efforts under the joint prevention and control mechanism, and guide public opinion positively and correctly; arrange news releases on the joint prevention and control mechanism, and where necessary, organize press conferences; track public opinion at home and abroad, and clarify facts in a timely fashion; strengthen management and guidance on the release of (continued) . the establishment, composition and operations … reports, discuss and address problems, and push ahead with prevention and control work within their fields. the establishment and improvement of the coordination mechanism remarkably increased the efficiency of inter-departmental coordination and response efforts. this success evinces the importance of a multi-departmental coordination and collaboration mechanism based on risk communication for effective epidemic prevention and control. thirdly, horizontal collaboration between related departments was strengthened. for example, on april th, , the moh office of health emergency sent a letter to the miit department of consumer goods industry recommending an increase in the national stockpile of supplies necessary for influenza a (h n ) prevention and control including medical supplies and response gear. in another example, the office of health emergency, department of medical affairs, and bureau of disease control and prevention of the moh, the china cdc, and the chinese medical association, jointly formulated the technical guidance on prevention and control of human-swine influenza, and the plan for diagnosis and treatment of human-swine influenza ( ). one more example occurred on july th, when the foreign collaboration group for the joint prevention and control mechanism, along with the ndrc, the miit, and the sfda met to discuss donating influenza a (h n ) prevention and control materials to the who and developing countries affected by the pandemic. during the course of the epidemic response efforts, risk evaluation, and risk management, it was necessary to build unobstructed information exchange channels to ensure the accurate transfer of data and information. under the joint prevention and control mechanism, each of the lead departments for the work groups and the expert advisory committee appointed people to collect, sort out, tabulate and analyze their work groups' epidemic information and latest progress on a daily basis, and to report in writing daily data collected by : to the general group prior to : p.m. the general group would then prioritize epidemic information and progress reports, and submit it representing the entire joint prevention and control mechanism to the general duty office of the state council. strengthened coordination and communication between the work groups and their members ensured an unobstructed flow of information as the working groups were informed of major issues as they happened. the general group was also charged with publishing information crucial for the public's knowledge on epidemic prevention and control. updates on influenza a (h n ) prevention and control were submitted via four reporting systems: the disease monitoring information reporting management subsystem of the china information system for disease control and prevention, the public health emergency reported information management system, the china influenza information monitoring system, and administrative reporting system for health departments. reported information mainly included: ongoing epidemic situations, monitoring results from sentinel hospitals and network laboratories, progress in vaccination and results of side effects monitoring, and ongoing regional and departmental response efforts. with influenza a (h n ) cases rising rapidly, issues with epidemic information reporting began to occur, such as overlapping reports, large discrepancies between confirmed reported cases and actual cases, and the circulation of ambiguous epidemic information. to better and more accurately reflect national epidemic situations and trends, the moh general office issued the notice on strengthening work of reporting deaths from influenza a (h n ) on november th, , and the notice on adjusting the work of reporting information on the influenza a (h n ) epidemic on november th, . by april th, , the general group had submitted more than work reports regarding influenza a (h n ), and compiled and published over response progress to influenza a (h n ) under the joint prevention and control mechanism reports. information on epidemic trends and response efforts was released in a timely, open, and transparent manner, and by this time eight news conferences and nine press briefings had been held regarding the latest progress in influenza a (h n ) prevention and control. to ensure that prevention and control measures were implemented effectively and efficiently, the work groups each established a supervision and inspection system by which to examine routine work on a regular basis, identify existing deficiencies and problems, and supervise and inspect response contingency plans and procedures, operational capacities, epidemic monitoring, epidemiological investigations, designated hospitals and their isolated areas, medical observation, material supplies, staff training, and information dissemination on prevention and control. in the course of influenza a(h n ) prevention and control efforts, local governments, as instructed by the central government, examined their own conditions and established local bodies to command and coordinate response measures. the local departments worked together to implement disease prevention and control measures in priority areas and among targeted groups, and ensured continued epidemic monitoring and treatment. the following three modes mainly represent actual prevention and control measures adopted by local governments. the first work mode was similar to the national joint prevention and control mechanism. for example, shaanxi set up a leading group for influenza a (h n ) prevention and control, whose office was located inside the provincial department of health and their local structure followed the " + " joint prevention and control mechanism model. guangdong established a joint prevention and control mechanism with the participation of thirty two departments, and nine work groups and three panels of clinical, disease prevention and control, and etiological experts functioned under the mechanism. on april th, fujian established an influenza a (h n ) prevention and control work group, headed by a provincial government official; the office was located inside the building of the provincial department of health whose emergency management office was charged with performing routine work for the group. the emergency guidance on influenza a (h n ) prevention and control in fujian (tentative), issued on may th, , outlined response guidance as "prevention first through joint prevention and control, timely management, and level-by-level responsibility." the second work mode was the emergency operations center or leading group. after discovering their first confirmed influenza a (h n ) case, some provinces and cities upgraded their existing disease prevention and control mechanisms and established an influenza a (h n ) response leading group or emergency operations center. beijing was the first in the country to establish a municipal-level public health emergency operations center in may , and had earlier (april th, ) launched a public health emergency response mechanism after the who declared the outbreak of swine influenza in mexico; an influenza prevention and control office (at the general office of the municipal government before it relocated to the municipal bureau of health) was established under the emergency operations center, whose members included twenty two committees, eighteen district and county governments, and the gld health department. this control office established a public health emergency response and medical rescue collaboration mechanism with the china cdc, the academy of military medical sciences, and other institutions. it established a mutual fixed epidemic communication system with local agricultural, educational, industrial, and commercial departments. a joint command response mechanism was also created with other special operations centers in beijing. on may th, shandong established a provincial public health emergency leading group and started level-ii response measures after discovering its first-and the country's second-confirmed imported case of influenza a (h n ). on june nd, hubei established an influenza a (h n ) emergency operations center after the province's first case was confirmed. immediately after the country's first influenza a (h n ) case was confirmed on may th in sichuan, sichuan initiated level-ii public health emergency response measures and set up a provincial response leading group as per the state council's requirement of handling the virus as a category b infectious disease. the third work mode was the joint conference system. some provinces and cities established a joint conference system in response to the influenza a (h n ) outbreak. for example, henan established an influenza a (h n ) joint conference system on april th, and on the same day guangxi established a -department joint conference system for its own prevention and control efforts. while establishing provincial-level joint prevention and control mechanisms, health departments also set up internal expert panels. for example, fujian provincial department of health set up a provincial-level influenza a (h n ) prevention and control expert supervision panel; guangdong provincial department of health established three expert panels for clinics, disease prevention and control, and etiology; sichuan provincial department of health established a leading group, a technical guidance expert panel, and a medical rescue panel on april th. the composition of local mechanisms for influenza a (h n ) prevention and control basically followed the framework of the national joint prevention and control mechanism, with an office and several work groups collaborating under a leading group or operations center. for example, beijing's public health emergency operations center was responsible for the city's influenza prevention and control, and it was comprised of eight work groups plus an office, these groups included: immigration inspection, healthcare, epidemiological survey, material security, dissemination and communication, information, animal husbandry and veterinary, and social prevention and control supervision (referred to as "one office and eight groups"). fujian's influenza a (h n ) prevention and control leading group consisted of thirty one departments and organizations, including the provincial department of health, a press office, and a development and reform commission. sichuan's influenza a (h n ) prevention and control leading group (operations center) included departments from emergency management, public security, development and reform, transportation, immigration inspection and quarantine, tourism, civil aviation, foreign affairs, and publicity. the office of sichuan's influenza a (h n ) prevention and control leading group was originally located in the provincial department of health, which was then moved to the provincial government's general office building as the epidemic worsened. its work groups consisted of emergency coordination, general support, information secretaries, epidemic prevention and control, medical rescue, supervision and inspection, press and communication, and health education. local influenza a (h n ) prevention and control mechanisms adopted a similar communication and coordination mechanism to the joint national prevention and control mechanism, and operated in with joint offices, conferences, etc. sichuan is one example of this. the provincial operations center and the provincial leading group (headquarters) shared offices in order to strengthen the province's joint prevention and control against influenza a (h n ), and its seven work groups were comprised of highly capable professionals from the emergency management office, third secretariat office of the general office, and the provincial health department. the health department met regularly with the departments of public security, civil aviation, immigration quarantine, economy and trade, animal husbandry, as well as pla and people's armed police troops stationed in the province. adjustments were made in real time in according with latest local epidemic situations. latest information on epidemic updates across the province were reported daily to members and related departments, and where cases were discovered, the departments of health, public security, foreign affairs, railway, transport, and others worked closely to track close contacts and ensure they were medically observed. through close collaboration between departments at various levels on joint prevention and control, sichuan ensured that cases were discovered, reported, isolated and treated at the earliest possible time, which delayed the spread of the virus and lowered epidemic intensity. government departments, enterprises, institutions, communities, and nonprofit organizations (npos) all play important roles in prevention and control of an infectious disease. with the country's response efforts entering its second phase, the th meeting of the national joint prevention and control mechanism, held on june th, , proposed further improvements of existing mechanisms, in particular establishing accountability systems and mass prevention and control mechanisms with participation from urban communities, schools, enterprises and villages. these mechanisms could better implement tailored measures, disseminate self-protection knowledge for families and individuals, and improve measures that maintain the status quo and normal economic operations. when confronted with a public health emergency, under the guidance of the government, the society can effectively avoid or reduce potential damage by achieving preliminary prevention and control targets at local levels through community involvement and solidarity along with raising public awareness in self-protection. in its work report, the who mentioned two types of community participation, i.e. participation as a means, and participation as a goal, and analyzed effects of the two. in the course of china's influenza a (h n ) prevention and control efforts, communities, the most basic social units, played an important role in knowledge dissemination and health education, the tracking and isolation of close contacts, and epidemic supervision. when uncertainties still surrounded the pathology and virulence of influenza a (h n ) in the early days of the epidemic, local communities launched information dissemination and health education campaigns, playing a crucial role in stabilizing public opinion and raising awareness of scientific disease prevention and treatment methods. in the case of community-level outbreaks, affected communities generally adopted comprehensive response measures, which emphasized managing the sources of infection in order to contain and control the transmission of the influenza virus. measures mainly included the following: ( ) sub-district offices or town governments mobilized social forces-as per laws, rules, and regulations-to provide support for isolated cases, including logistical service to personnel engaging in medical observation; ( ) close contacts were medically observed centrally or at home, and healthcare workers reported daily on patients' progress; ( ) patients with influenza-like symptoms were recommended to rest at home and not participate in unnecessary public gatherings or travel; ( ) schools, nurseries and kindergartens, nursing homes, and construction sites were required to conduct health inspections, and enterprises with a concentrated amount of personnel or those who provided social services were required to perform morning health inspections; ( ) information on epidemic trends and response measures were published in real time, and efforts were made to strengthen information disclosure within communities; and ( ) health education and risk communication were carried out through multiple channels. when the virus broke out in communities, healthcare departments managed cases categorically and adopted comprehensive measures for strengthening the treatment of severely ill cases, lowering case fatality rates, and mitigating epidemic damage. with prevention and control measures in place against community-level outbreaks, response measures for priority areas mainly included the following: ( ) as per related laws, rules, and regulations, local governments mobilized social forces to ensure the logistical support of measures like home-based treatment of cases with influenza-like symptoms; ( ) migration was cut or restricted, recreational areas were temporarily shut down, and large-scale gatherings were canceled or postponed. enterprises and institutions within communities were permitted to grant time off for all or some of their employees; ( ) schools, nurseries, and kindergartens were closed per related regulations; ( ) enterprises and institutions as social services providers with large workforces implemented a health reporting system, management was enhanced where there were large flows of people, and people with influenza-like symptoms were recommended to rest and receive treatment at home; ( ) when necessary, outbreak points were put under isolated control, and quarantine measures were taken in epidemic areas. at the same time, measures were taken to organize and encourage volunteers to participate in prevention and control activities, to help maintain the normal operation within communities, and provide mental health interventions to avoid adverse effects on public health. these measures, which were designed based on real community conditions, effectively guaranteed the protection of the status quo, and laid a strong foundation for local influenza a (h n ) prevention and control efforts. in the course of influenza a (h n ) prevention and control efforts, drug stockpiling enterprises responded actively to the government's call for material reserves and production. because influenza drugs weren't prevalent in clinical use, they were traditionally stockpiled through loans, government subsidies, business opportunities and moderate enterprise compensation. problems arose during the implementation of this mechanism such as subsidy inaccessibility and unreasonable compensation. for example, the current % subsidy policies regarding corporate loans and government subsidies hardly met the needs of enterprises, and the problem of unreasonable compensation to pharmaceutical enterprises still existed, partly because specific mechanisms were lacking. at the same time, more than % of emergency response drugs were not on the standing list of medications, and so it was necessary to build a long-term relationship between the government and enterprises to specify respective duties, and link stockpile funding with corporate social responsibility to balance compensation. reagent manufacturing was one of the government's top priorities during the entire course of its influenza a (h n ) prevention and control efforts. some reagent manufacturers which had developed and produced reagents for biological agents such as anthrax during the olympic games already had experience in emergency response. for example, beijing kinghawk pharmaceutical co., ltd., the country's first to obtain approval for an influenza a (h n ) testing kit, signed a strategic alliance agreement with the china cdc during the influenza a (h n ) epidemic. the enterprise also provided its laboratories voluntarily when there was no clear policy on state funding, doing its best for society as a corporate citizen. with its technology reserve, seven production platforms and ninety approved products, kinghawk was able to perform research and development on product standardization during a critical time of the epidemic. the china cdc had access to international resources for preliminary research and development and successfully obtained information and strains from the who. this collaboration between the two parties made it possible to develop preliminary products in h and thus ensured that considerable demand for clinical diagnosis was met. this played a positive role in case diagnosis during the early phases of the influenza a (h n ) epidemic and was also quite meaningful in terms of drug use guidance. the government provided active support to research and development efforts. take kinghawk as an example. after kinghawk signed the agreement with the government on may th, , both the beijing economic-technological development food and drug administration and the beijing food and drug administration provided recommendations. kinghawk had developed a rapid test kit by may th, received approval from the cfda on june th to launch the emergency response system, and got approved for manufacturing the reagent for , people on september th. kinghawk had collaborated with the china cdc in the past and had experience in reagent development and manufacturing. the development of the reagent fully demonstrated the efficiency in collaboration between the government and a commercial enterprise, and also guaranteed the timeliness of preliminary disease diagnosis. in the containment phase, hotels and other requisitioned enterprises across the country showed full support for the response measures by providing isolation zones of influenza a (h n ) cases. many hotels suitable for isolation purposes were private firms and thus could not be requisitioned through administrative orders, which put a certain amount of pressure on local governments. however, coordination efforts by local governments did earn support and assistance from these hotels. transport enterprises shouldered the heavy task of implementing disease prevention and control for the floating population. civil aviation, railways, road and related enterprises implemented strict prevention and control measures, including disseminating knowledge about disease prevention and control, and providing necessary infrastructure support for emergency response efforts targeting the floating population. all in all, a healthy transportation environment helped lower the transmission of the disease. facing the unexpected onset of influenza a (h n ), these transportation enterprises all established prevention and control groups. for example, the caac north china regional administration established a capital airport influenza a (h n ) prevention and control leading group, which was based on the former capital airport public health emergency leading group; beijing capital international airport company limited, air china limited, china southern beijing company, china eastern beijing company, hainan airlines beijing company, caac air traffic management bureau all set up their own epidemic response teams to ensure the orderly implementation of prevention and control measures. telecommunications enterprises did their duties as corporate citizens and actively cooperated in influenza a (h n ) prevention and control efforts. china mobile group beijing company limited, china telecom group beijing company limited, china unicom beijing company limited, among other telecommunications operators, suspended their normal user notification group-messaging services and mustered network resources to send messages on epidemic updates while increasing maintenance staff, strengthening network monitoring, and closely watching the impact of group messaging on their systems. nonprofit organizations, or npos, are organizations that fulfill particular social causes or missions without seeking profit for their efforts, and npos represented a crucial social force in the course of the nation's influenza a (h n ) prevention and control. for example, the beijing red cross established a public health emergency operations center which consisted of a general information group, a rescue response group, a fundraising and aid group, a publicity group, and a public relations group. this organization actively engaged in response efforts in accordance with the beijing red cross emergency contingency plans for public emergencies. the entire nation was involved in epidemic prevention and control, including its citizens. during the response to the virus, volunteers played an important role when multiple departments suffered emergency manpower shortages. for example, medical and healthcare students in colleges and universities volunteered to work on the front lines of epidemic prevention and control. in beijing, student volunteers from capital medical university assisted with response efforts at capital airport, and similar volunteering also occurred in fujian. the public actively supported the government's influenza a (h n ) prevention and control measures, and voluntarily took part in the process via the internet and other media channels; for example volunteers called those who had just returned from abroad and informed them of potential isolation measures. at the same time, increased public health awareness was also instrumental in successfully dealing with the disease. prevention and control mechanisms at the beginning of the influenza a (h n ) epidemic, china established a national level emergency management mechanism directly under the leadership of the state council that enabled cross-departmental joint prevention and control collaboration, which provided an effective organizational support and operation mechanism for the response efforts. though the moh had formulated the ministry of health's influenza pandemic preparedness and response plan (tentative) before the epidemic broke out, this document focused only on the duties of the moh and didn't encompass more complex coordination and collaboration with related government departments. the joint prevention and control mechanism remedied this flaw by providing a platform for coordination and collaboration between the moh and other related departments. also, because this mechanism was not like the state council's operations center, it allowed some space for strengthening the state council's leadership and collaboration once the epidemic worsened. during the prevention and control efforts, local governments adapted and innovated central policies and their implementation in light of local epidemic situations, public health trends, and demographic and economic conditions. some areas established prevention and control mechanisms with local characteristics. the main features of these mechanisms are as follows: the first was the establishment of a strong leadership system. in the process of prevention and control, local governments established their respective public health emergency leadership systems based on local epidemic situations, geographic features, and public health resources. the second was the innovation in ideas and methods. local epidemic prevention and control bodies closely monitored trends and reengineered their methods based on existing departmental systems in order to better target obstacles encountered in operations. for example, in the early days of the epidemic, the beijing government issued a notice on further specifying duties and prioritizing operations to strengthen influenza a (h n ) prevention and control, which articulated the new public health notion of "responsibility of four sides" (government, departments, enterprises, and individuals) . this clarification brought about effective collaboration between the government and the society in public health emergency management. the beijing immigration inspection and quarantine bureau employed risk analysis methods in its prevention and control efforts and ensured electronic transfer of information on inbound passengers, which not only increased quarantine and inspection efficiency but also scientifically and efficiently improved response measures. fujian was the country's first province to implement temporary isolation measures through its local health department. henan created an epidemic prevention and control network of "three horizontal fronts"-arrangements at a government level, measures at enterprise (institution) level, and protection at a local level; and "three vertical fronts"-government supervision, inter-departmental collaboration, and public opinion guidance. these institutional innovations proved very effective in the response efforts. the third was the establishment of an inter-provincial support mechanism. on november th, , the moh general office issued the notice on strengthening medical treatment of influenza a (h n ) patients (no. , ) , announcing the decision to establish an inter-provincial support mechanism for medical treatment of influenza a (h n ) patients as per the notice of the state council on strengthening the ongoing work on influenza a (h n ) prevention and control (no. , ) and as needed for patients. the form of assistance was technical support, especially in regards to medical treatment of seriously and critically ill patients. throughout the entire duration of the prevention and control efforts, governments greatly heeded experts in various fields, which aided governments in creating more scientific policy adjustments and technical plans, and consequently reduced blindness and uncertainty in policy implementation. experts from cdcs, hospitals, publicity departments, and other departments took part in the decision-making process, and their input was adopted in real time. some experts even took the initiative to provide police recommendations directly to decision makers. at the same time, governments sought out expert opinions through different methods and channels, i.e., consultation at joint prevention and control meetings or direct consultation with the experts. expert recommendations ensured scientific policies and more targeted and effective policy formulation. in regards to policymaking, some local governments formulated policies and adjustments based on local conditions and epidemic trends. for example, jiangmen experimented with a home-based isolation policy, while shenzhen created corridors at ports specifically for foreigners and a separate one for students commuting between shenzhen and hong kong for school. also, in terms of policy adjustment, some local departments were able to adjust related policies in time to better suit local epidemic situations. as for issues that necessitated policy coordination, local departments also made strategic adjustments as early as possible. for example, the guangdong immigration inspection and quarantine bureau, at experts' suggestion, transferred persons who required isolation and medical observation to health departments for categorical management, which thus ensured the efficient use of epidemic prevention and control resources. over the course of influenza a (h n ) prevention and control efforts, the government cultivated an environment of widespread social participation under the leadership of the party and government, with enterprises, communities, volunteers and other social actors playing crucial roles in the response efforts. the joint national prevention and control mechanism was essentially a command and decision-making mechanism established according to the potential amount of damage influenza a (h n ) could inflict upon society. on the one hand, influenza a (h n ) response required inter-departmental collaboration, and relying solely upon health departments for countermeasures wouldn't be enough; on the other hand, because the virus was not as virulent as to merit the establishment of a state council operations center (or headquarters), the state council instead instructed the moh to establish a multi-departmental joint prevention and control mechanism; and this new organization represented a relatively flexible and effective response mechanism. although local governments were already aware of the epidemic at its onset and were actively engaging with different departments in their response efforts, because there were no explicit provisions in related laws and contingency plans for the joint national prevention and control mechanism at the central level, no corresponding normative documents were available for its implementation at a local level. no unified standards on the name, content, form of establishment, and system structure for local governments' prevention and control bodies existed. although local governments adapted as they went, it was still an environment that incited disorder and confusion. on the one hand, participating departments fully endorsed the joint national prevention and control mechanism. this mechanism, they thought, possessed several advantages: firstly, the joint consultation system made it possible to directly formulate and sign policies at joint prevention and control conferences, which saved time for everyone; secondly, the joint briefing system required the work groups to send daily reports to other units and departments, thus facilitating both inter-group and inter-departmental communication; and finally, internal collaboration within groups was solid, and the briefing system allowed an unobstructed flow of information. however, the joint national prevention and control mechanism based upon consultation and communication had its limitations. on issues involving departmental interest, division of duty, and so on, this horizontal collaboration was less efficient than regulation and control by a single, high level leadership department. one contested issue dealt with the location of the local joint prevention and control office: should it be set up in the comprehensive emergency management office of the local government or in the emergency management office of a local specialized department. some provincial emergency management offices insisted that for an emergency event like the ongoing influenza a (h n ) epidemic, a joint prevention and control office should be located in a specialized department so as to leverage the department's expertise and increase response flexibility, convenience and efficiency. in this scenario, the provincial emergency management office would be tasked with solving issues that the specialized department could not. on the other hand, some provincial health department's emergency offices argued that if the office was located in the local government, the joint prevention and control office would enjoy greater authority and more efficient collaboration. achieving a smooth and effective transition between peacetime and public emergency, and establishing mechanisms that combined crucial components from both systems, was a new challenge that arose in the influenza a (h n ) epidemic. after the sars epidemic, local governments established permanent public health emergency response departments and corresponding working mechanisms to deal with future public health emergencies. these departments and mechanisms should have been employed upon the onset of the influenza a (h n ) epidemic. however, most provinces established completely new leading groups only after the central government established joint national prevention and control mechanism. in one example, a provincial health department already had a permanent public health emergency operations center, but, after the central government established the influenza a (h n ) joint national prevention and control mechanism, this province created an entirely new prevention and control leading group and a port leading group. at the same time, the health department also established new eternal mechanisms, including: the provincial cdc established an emergency response department with leaders from major sections like emergency management and vaccination planning (starting in , this provincial cdc implemented a " in " meeting system with participation from emergency management, disease control, and the disease monitoring department). the main reason for this redundancy was because the central government did not provide specific conditions or qualifications for contingency planning and management for the transition period between peacetime to emergency. thus, local governments lacked a clear transition mechanism that they could utilize. it was the reason that many local governments chose to re-establish emergency management bodies when influenza a (h n ) broke out. as public health emergency management involved multiple collaboration systems from the central government down to local governments, regions, and departments, inter-departmental collaboration in the response efforts was intrinsically complicated. the response to this epidemic revealed problems that existed both in horizontal and vertical coordination. in regards to horizontal coordination between central departments, the health, education, security, transportation and many other departments were involved in the influenza a (h n ) response efforts, which created an environment where responsibilities could easily overlap and grey areas would occur in management. there was also a lack of coordination and standardization between central-level ministries' policy documents for influenza a (h n ) countermeasures. for example, in regards to content standardization, the health authorities felt that using the temperature of . °c as the sole standard for sending people to the hospital was unreasonable and would cause an unnecessary burden on hospitals. in regards to time standardization, on december nd, , one province stipulated that only patients with a temperature of °c or higher must be sent to a hospital, and it took the country two more weeks to follow suit. in regards to inter-departmental work, port laboratories in some provinces had begun testing in the early days of epidemic, but stopped after provincial health departments decided that ports were not fit for such work. obstacles also arose in horizontal coordination and collaboration between local departments. a lack of information communication between local departments due to the unavailability of complete information in the early stages of prevention and control made it nearly impossible for effective collaboration. in regards to the division of labor and coordination between the central and local governments for disease prevention and control, some local governments held that the central government should have presented broader goals and authorized provinces and cities greater autonomy in their response measures. some felt that the central government should not have made influenza a (h n ) prevention and control an issue of political significance but should have been objective in understanding the differences between executive leadership and scientists' opinions. while the main duty of administrative leaders should have been to organize and mobilize social resources needed to cope with the epidemic, scientists should have been the ones to handle technical issues such as epidemic analysis and response measures. at the same time, more efficient communication should have been present between central and local departments tasked with specific operations. for example, some local management departments felt that the entire process was quite political, making some documents difficult to fully implement; in the two most volatile months that lasted from april th to june, documents were issued frequently, and in some cases were in conflict with one another and lacked integrity and continuity. in regards to adjustment of prevention and control strategies, some regions' health departments reported the following issues: higher-level departments frequently adjusted technical guidance and strategies for prevention and control, there was a wide variety of information reporting methods and they were constantly in flux, different departments formulated their own response requirements, and differences occurred in measures and standards; all of which greatly complicated local response operations. certain communication and coordination issues also existed within the health department's internal system. the moh internal horizontal collaboration needs to be strengthened epidemiological investigations, clinical diagnoses, and laboratory testing to combine the medical treatment and disease prevention. for example, the china cdc played a crucial role as a central technical support body of influenza a (h n ) prevention and control in epidemiological information collection, monitoring, analysis, and judgment, but at the same time it also had a lot of administrative duties, and its services and duties overlapped with those of the moh's bureau of disease control and prevention. there should be unified leadership and coordination between higher and lower-level health departments within the national epidemic prevention and control system. a certain degree of flexibility is also necessary as provinces differ in epidemic situations, medical resources, geographic features, and so on. in regards to information reporting within the health system, though the china cdc and the moh had established information systems relating to epidemic surveillance, including an epidemic direct reporting system, no information sharing mechanism was created between the china cdc and medical institutions; in particular, some county level medical institutions didn't even have sound data collection and reporting systems. this resulted in a single point of decision making and command, and their lack of network and information technology weakened the support they could've had in implementing response measures. npos such as the red cross society of china played important roles during the influenza a (h n ) prevention and control efforts. however, by comparison with developed countries, china still lags behind in terms of public participation in public health emergencies. there still remain limitations in skill and knowledge, as no emergency volunteer systems or working mechanisms were formed, and no leveraging of npo resources really occurred. on public emergency management bodies of the chinese government key: cord- -psnec qp authors: mbareche, hamza; veillette, marc; pilote, jonathan; létourneau, valérie; duchaine, caroline title: bioaerosols play a major role in the nasopharyngeal microbiota content in agricultural environment date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: psnec qp background: bioaerosols are a major concern for public health and sampling for exposure assessment purposes is challenging. the nasopharyngeal region could be a potent carrier of long-term bioaerosol exposure agents. this study aimed to evaluate the correlation between nasopharyngeal bacterial flora of swine workers and the swine barns bioaerosol biodiversity. methods: air samples from eight swine barns as well as nasopharyngeal swabs from pig workers (n = ) and from a non-exposed control group (n = ) were sequenced using s rrna gene high-throughput sequencing. wastewater treatment plants were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. results: a multivariate analysis showed air samples and nasopharyngeal flora of pig workers cluster together, compared to the non-exposed control group. the significance was confirmed with the permanova statistical test (p-value of . ). unlike the farm environment, nasopharynx samples from wastewater workers did not cluster with air samples from wastewater treatment plants. the difference in the microbial community of nasopharynx of swine workers and a control group suggest that swine workers are carriers of germs found in bioaerosols. conclusion: nasopharynx sampling and microbiota could be used as a proxy of air sampling for exposure assessment studies or for the determination of exposure markers in highly contaminated agricultural environments. the microbial flora of aerosols, referred to as bioaerosols, consists of a combination of viable and non-viable microorganisms (e.g., bacteria, fungi and viruses) and derived compounds of biological origin (e.g., animal and plant debris, endotoxins, exotoxins, and other microbial metabolites) [ ] [ ] [ ] . bioaerosols are ubiquitous in indoor and outdoor environments and are generated from various natural and/or anthropogenic sources. composed of particles ranging in size from a few nanometers to µm in diameter, bioaerosols remain suspended in the air for long periods of time and may travel many kilometers depending on the size of the particle [ , [ ] [ ] [ ] [ ] [ ] . therefore, the dispersal of bioaerosols may impact the air quality of extensive areas that are far from the source and can create public health issues, due to the presence of highly diverse and dynamic microbial communities. beyond affecting the time that particles remain suspended and the distances they travel, particle size plays a role in human diseases, as it dictates which pathway the particle follows in the respiratory tract after inhalation [ ] . for example, particles with a size of µm to µm tend to get deposited in the upper airways, while larger particles may remain in the nasal cavity [ , ] . bioaerosols can be a transmission vector for infectious diseases and are responsible for a variety of health problems, principally through inhalation [ , [ ] [ ] [ ] [ ] . human exposure to bioaerosols is associated with a wide variety of acute and chronic diseases ranging from allergies, asthma, rhinitis, sinusitis and bronchitis, mostly due to occupational exposure [ , [ ] [ ] [ ] [ ] . however, health risks from bioaerosols also exist just from living in close proximity to an intensive source of airborne biological particles [ , ] . additionally, other health problems linked to bioaerosols include fatigue, headache, mucous membrane irritation syndrome, nasal congestion, sore throat, and irritation of the nose and eyes [ , , ] . the industrial environment is the main source of occupational health issues, due to the presence of raw organic materials, the prevalence of operations releasing harmful bioaerosols (e.g., mechanical operations such as wood planning, straw chopping, animal bedding, hay handling, and compost pile turning) and the eventual large amounts of bioaerosols present in confined spaces. for example, biowaste facilities are characterized by notable concentrations of bioaerosols, due to the intense microbial activity involved in waste degradation and the activities performed by workers [ ] [ ] [ ] [ ] . wastewater treatment plants (wwtps) represent another environment where workers are subject to bioaerosol exposure, due to the steps required for the treatment of discharged municipal and industrial effluents [ , ] . intensive animal farming practices in confined buildings that hold a large number of animals (e.g., pigs, poultry, cattle) are also associated with extreme exposure to airborne microbes. the variety of possible sources (e.g., animals, feces, feed, litter) present at farms leads to the emission of complex mixtures of biological particles [ ] [ ] [ ] [ ] [ ] [ ] . moreover, the dynamic nature of the microbial composition makes health risk evaluation complicated for farm workers and nearby residents [ , ] . environmental hygienists continue to insist that insufficient exposure assessments are a primary reason for the absence of bioaerosol exposure limits and strategies to mitigate risk [ , ] . there are several challenges and limitations to measuring bioaerosol exposure. the type of sampler, time and duration of sampling, meteorological conditions [ ] , and geographical positions all affect bioaerosol sampling efficiency, making it difficult to compare studies and limiting collaboration efforts in bioaerosol exposure studies. an added challenge in terms of measuring microbial diversity is that culture-dependent analytical approaches recuperate the cultural/viable portion of bioaerosols exclusively. in contrast, high-throughput sequencing (hts) methods are associated with a more in-depth characterization of the microbial content of a sample [ , , [ ] [ ] [ ] [ ] . to overcome the aforementioned challenges, the scientific community that studies aerosols has identified the need to explore new alternatives and complementary approaches for assessing bioaerosol exposure, such as identifying markers that can help classify environments based on human health risks [ ] [ ] [ ] . the upper respiratory tract, which includes the nasal cavity and the nasopharynx and is a primary pathway for inhaled air, is an important niche for transient environmental bacteria and for colonization. previous studies have already used nasopharynx or nasal cavity samples to look for specific microorganisms, using culture-based approaches [ , ] , or revealed the presence of bacterial resistance genes, using molecular biology approaches [ ] . recently, an hts approach was used on samples from the anterior and posterior cavity of the nose to study the bacterial diversity of individuals [ ] . however, because the microflora in the nasal cavity is dynamic and fluctuant [ ] , a region like the nasopharynx may represent a more long-term reservoir of inhaled bioaerosols. to the best of our knowledge, no study has ever used a s rrna amplicon-based hts approach to investigate the microbial diversity of nasopharyngeal flora, in order to assess occupational exposure. microbiome studies that use upper respiratory tract tissue and focus on specific pathologies (such as asthma, chronic obstructive pulmonary disease and chronic rhinosinusitis) may underestimate the effect of occupational exposure on the microbiomes of the subjects examined in those studies. we hypothesis that the microbiome of patients with respiratory disease is most likely affected by exposure to their work environment in addition to disease, rather than to disease alone. this study aims to contribute to the development of a new alternative method for assessing bioaerosol exposure, by linking the nasopharyngeal flora of pig farm workers to the bioaerosol microbial composition of their occupational environment, and to explore the role of bioaerosols in the nasopharyngeal microbial content. the s rrna amplicon-based hts approach was used to determine bacterial diversity, while qpcr was used to evaluate the presence of human pathogenic agents and bacterial resistance genes in bioaerosols and in nasopharyngeal samples from pig workers. additionally, wwtps were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. although the experiment focused on pig farmers and the buildings that they work in, the results may have implications for a wider population of agricultural workers. consequently, nasopharynx samples could be used as proxies for air samples in exposure assessment studies and for the determination of exposure markers in agricultural settings. in addition, the study advocates for the need of systematic bioaerosol exposure study when evaluating the nasopharyngeal microbiota. air samples were collected from eight confined pig buildings in eastern canada during fall/winter . inside each farm building, a sampling site was designated based on worker activities and bioaerosol exposure. the buildings visited were mechanically ventilated and contained between - pigs each weighing between - kg. there were no obvious signs of illness affecting the animals. air samples were collected from eight wwtps in the province of quebec, located in eastern canada, the during summer and winter seasons. summer visits occurred between september and july , and winter visits occurred between february and march . four sampling sites were chosen depending on the wastewater treatment process (screening, de-gritting/degreasing, settling tank, and bio-filtration), workers' daily tasks, and the level of confinement of the space. a liquid cyclonic impactor coriolis µ biological air sampler (bertin corp., rockville, md, usa) was used for collecting air samples. the samplers were set at l/min for min ( m of air per sample), placed within - m of the bioaerosol source and away from any turbulent air flow (e.g., away from building exhaust fans). fifteen milliliters of a phosphate buffer saline (pbs) solution ( . m, ph . , lonza, walkersville, md, usa) were used as the collecting solution. nasopharyngeal samples were taken from controls (university students and staff never exposed to animal farms), pig farmers and wwtp workers between the fall of and spring . all controls, pig farmers, and wwtp workers were non-smokers and none were taking antibiotics. the protocol was approved by the ethics committee of the institut universitaire de cardiologie et de pneumologie de québec (cer ). nasopharyngeal samples were collected by a nurse using swabs (puritan ® hydraflock ® collection devices, guilford, ma, usa) with a dry secure transport system. briefly, a swab was inserted into the nose until a resistance was felt and then turned a few times before it was removed. samples were transported to the laboratory at • c. from the ml collecting solution of the coriolis µ biological air sampler (bertin corp.), a . ml aliquot was centrifuged for min at , × g (j. pilote protocol = ml aliquot, min, , × g). the supernatant was discarded and the pellets were kept at − • c until dna extraction. likewise, the tips of the swabs were cut and vortexed thoroughly in ml pbs (lonza) and discarded. suspensions were then centrifuged for min at , × g, the supernatants were discarded and the pellets were stored at − • c until dna extraction. total genomic dna from air and nasopharyngeal samples was extracted with a powerlyser ® powersoil isolation dna kit (mo bio laboratories, carlsbad, ca, usa) following the manufacturer's instructions. dna samples were stored at − • c until subsequent analyses. the amplification of targeted genes, equimolar pooling, and sequencing were performed at the plateforme d'analyses génomiques (ibis, université laval, québec, qc, canada). the s rrna v -v region was amplified using the sequence-specific regions described in comeau et al. using a two-step dual-indexed pcr approach, specifically designed for illumina ® instruments, san diego, ca, usa [ ] . the gene-specific sequence was first fused to the illumina ® truseq sequencing primers and pcr was carried out in a total volume of µl containing × q buffer (neb, ipswish, ma, usa), . µm of each primer, µm of each of the dntps, u of q high-fidelity dna polymerase (neb) and µl of template dna. pcr thermoprotocol began with an initial denaturation at • c for s followed by cycles of denaturation at • c for s, annealing at • c for s, extension at • c for s and a final extension at • c for min. the pcr reaction was purified using the axygen pcr cleanup kit (axygen ® , waltham, ma, usa). the quality of the purified pcr product was checked on a % agarose gel. a fifty to -fold dilution of the purified product was used as a template for a second round of pcr in order to add barcodes (dual-indexed) and for missing sequences required for illumina sequencing. the thermoprotocol for the second pcr was identical to the first one but with cycles. pcr reactions were purified again in the same way as above, checked for quality on a dna bioanalyzer chip (agilent ® , santa clara, ca, usa) and then quantified spectrophotometrically with the nanodrop ® (thermo fisher scientific, waltham, ma, usa). barcoded amplicons were pooled in equimolar concentrations for sequencing on the illumina ® miseq machine. the oligonucleotide sequences that were used for pcr amplification are presented in table . briefly, after de-multiplexing the raw fastq files, the reads generated from the paired end sequencing were combined using the make.contigs script from mothur [ ] . quality filtering was also performed with mothur, using the screen.seqs script to discard homopolymers, reads with ambiguous sequences, and reads with suspiciously short lengths. similar sequences were gathered together in order to reduce the computational burden, and the number of copies of the same sequence was displayed to monitor the abundance of each sequence. this de-replication step was performed with vsearch [ ] . the sequences were then aligned with the bacterial reference silva core alignment using the qiime script align_seqs.py [ ] . operational taxonomic units (otus), with a % similarity cut-off, were clustered using the uparse method implemented in vsearch. uchime was used to identify and remove the chimeric sequences [ ] . qiime was used to assign taxonomy to otus based on the silva database reference training dataset for taxonomic assignment and to generate an otu table. a metadata-mapping file was produced that includes information about air and nasopharyngeal samples. the microbial diversity analyses, including statistical analyses, conducted in this study, were achieved using qiime plugins in version . . as described in qiime scripts (http://qiime.org/scripts/). the names of the scripts used are mentioned in the results section of each analysis. pcr was performed with cfx- and cfx- touch™ real-time pcr detection systems (bio-rad laboratories, mississauga, on, canada) to evaluate the presence of six human pathogens (clostridium difficile, listeria monocytogenes, mycobacterium avium, salmonella spp., staphylococcus aureus, and methicillin-resistant staphylococcus aureus (mrsa)), and antibiotic and metal resistance genes (cephalosporin, colistin, zinc). the pcr mixture contained µl of dna template, - nm for each primer, - nm probe and µl of × iq™ supermix or iq™ sybr ® green supermix (bio-rad laboratories) in a µl reaction mixture. the results were analyzed using bio-rad cfx manager software, version . (bio-rad laboratories). positive control and standard curves ranging from × to one copy of the targeted genes were used for each protocol using genomic dna or synthetic genes as templates (integrated dna technologies, coralville, ia, usa). negative controls were included in the plates as ntc (non-template controls). all primers and probes were purchased from integrated dna technologies. the primers, probes, hybridization temperatures, amplicon sizes and original references for all targeted genes are listed in pilote et al. [ ] . for alpha diversity measures, the normality was verified using the d'agostino and pearson omnibus normality test. the assumption of data normality was not fulfilled. non-parametric mann-whitney u tests (two-tailed) were performed to highlight that there are significant differences in diversity measures between the groups of samples. a p-value ≤ . was considered statistically significant. all of the results were analyzed using the software graphpad prism . (graphpad software, inc., san diego, ca, usa). to determine the statistical significance of the variation in the observed microbial community composition with multivariate analyses (pcoa), a permanova test was performed on the unweighted unifrac matrix. the compare_categories.py qiime script was used to generate the statistical results. because permanova is a non-parametric test, significance is determined through permutations. in this case, permutations were used. a p-value ≤ . was considered to be statistically significant. detailed information about the performance of the test is presented in the multivariate section of the results. the non-parametric mann-whitney u test was used to ascertain whether or not differences in otu abundances were statistically significant between the controls and pig farmers. to test otu differential abundance, the null hypothesis was that the populations that the two groups of samples were collected from have equal means. the range of p-values obtained for the most differentially abundant otus between the control samples and the pig farmer samples are presented in the differential abundance section of the results. this study used both positive and negative controls. the negative controls include unused swabs that underwent the same extraction protocol as the swabs collected from the subjects of this study. a pcr amplification targeting the s rrna genes allowed us to confirm the very low biomass of the negative controls compared to the nasopharyngeal swabs from the pig workers and non-exposed controls. for this reason, negative controls did not pass the next step of illumina hts. additional negative controls consisted of outdoor air samples that were taken outside the pig buildings sampled in this study. these samples showed enough concentration of bacterial biomass with the pcr amplification. thus, outdoor negative controls were sequenced. however, the number of reads and subsequent otu clustering was low compared to the indoor air samples. during the rarefaction step, the negative controls were not included in the analyses due to a low number of sequences. the goal is to have a number of sequences per sample deep enough to cover most of the bacterial diversity. positive controls consisted of a mock community containing equal concentrations of bacteria purchased from atcc ( strain even mix genomic material atcc ® msa- tm ). sequencing of the mock community showed a taxonomic profile resembling the expected microbial community, but with different relative abundances. in total, air samples from pig buildings, nasopharynx samples from farmers and nasopharynx samples from the non-exposed control group resulted in , , sequences (air samples = , ; farmers = , , , controls = , , ). following quality filtering and the discarding of singletons, , , unique sequences clustered into otus. representing the non-agricultural control environment (wwtps), , , sequences came from air samples ( , ) and nasopharynx samples ( , , ) from plant workers. after quality filtering and the removal of singletons, , unique sequences clustered into otus. a rarefaction analysis was performed to validate the sequencing depth and to confirm the effective sampling of the microbial diversity using the alpha_rarefaction.py qiime script. the lowest-depth sample parameter was used for the rarefaction analyses, allowing equal numbers of sequences for all samples. therefore, the samples with a sequencing depth lower than the reference sample were excluded from the analyses. the higher the sequencing depth, the more likely that the full diversity coverage is attained. the sequencing depth was , sequences for all the groups of samples: the air samples from pig buildings, the nasopharyngeal samples of pig farmers and non-exposed controls. the points shown in figure were calculated as follows: ten values from to , analyzed sequences were randomly selected. for each of these values, the corresponding number of otus observed, was noted for all of the samples. then, the average number of otus observed, plus or minus one standard deviation, were calculated for each of the ten values. the samples were divided into three groups: air from pig buildings, pig farmers and non-exposed controls. the slope of the curves shows sufficient sequencing depth and good bacterial coverage in all samples. moreover, pig farmers and air samples showed the highest average number of otus compared to non-exposed controls. four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, nonexposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, non-exposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b) . the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary material). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less figure . taxonomic profile showing the relative abundance of each bacterial class across nasopharyngeal flora samples from pig farmers, non-exposed controls and air samples from pig buildings. taxa written in bold type were specific to farmers and air from pig buildings. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary materials). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in nonexposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in non-exposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. listeria monocytogenes was detected in % of non-exposed controls and in % of pig worker samples. mycobacterium avium was not detected in the nasopharynx samples of either group. likewise, antibiotic and zinc resistance genes were present at a higher frequency among pig farmers compared to non-exposed controls. moreover, cephalosporin, and colistin resistance genes were exclusively detected in the nasopharyngeal flora of pig farmers. table . human pathogens and antibiotic and zinc resistance genes in the nasopharyngeal flora of pig workers compared to non-exposed controls. given the many potential microbial sources, animal farmers inhale a variety of aerosolized bacteria that impact their health [ , , ] . in this study, the bacterial populations in bioaerosols from pig buildings were compared to those of the nasopharyngeal flora of farmers using bioinformatics tools to determine if nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. systemic microbial ecology analyses led to unequivocal results with identical conclusions throughout the analyses. the alpha diversity of bacterial species in the air from pig buildings and the nasopharyngeal flora of farmers were not statistically different. the evaluation of species diversity was introduced by whittaker and defined as the number of species and their proportional abundance within one sampling site [ ] . there are different ways to measure alpha diversity and an extensive list of indexes has been presented by magurran and mcgill [ ] . in addition to the usual chao richness estimates and shannon/simpson diversity measures [ ] [ ] [ ] [ ] , pd whole tree was also used to analyze the alpha diversity in samples in this study. pd stands for phylogenetic diversity and is defined as the minimum length of all phylogenetic branches required to span a given set of taxa on the phylogenetic tree [ ] . all four of the alpha diversity measures revealed greater bacterial richness and diversity in the nasopharyngeal samples from pig farmers compared to non-exposed individuals. the observed similarity between bioaerosols from pig buildings and the nasopharyngeal flora from farmers is indicative of occupational exposure and, consequently, a transient presence and/or possible colonization of the upper respiratory tract regions by environmental bacteria. these findings are even more interesting given that the majority of pig farmers recruited for this study do not work in the eight pig buildings selected for air analysis. this suggests that airborne bacteria associated with pig buildings can take over the microbiota in farmers' nasopharynxes. the establishment of this "new" microbial community could represent a microbial signature for the nasopharynx of pig farmers. also, a higher prevalence of viruses in the nasopharynxes of farmers compared to the non-exposed control group could play a role in the increased alpha-diversity [ ] . beta diversity analyses revealed that long-term exposure, such as occupational exposure to bioaerosols in the air of pig buildings, appeared to modify the nasopharyngeal microbiota of farmers. common approaches to evaluating changes in the community composition (beta diversity) rely on the creation of a (dis)similarity matrix to calculate the distance between samples. dis(similarity) matrices may be calculated using different methods depending on the type of dataset, analyses, and the objectives of the study, as some metrics are more suitable than others [ ] [ ] [ ] [ ] . the unifrac distance metric was used as efficacy was proven with s rrna bacterial genes [ ] . in addition, pcoa coupled with permanova offers a robust statistical significance of sample grouping using distance matrices. this non-parametric multivariate analysis of variance separates the distance matrix into sources of variation to describe the robustness and significance of a variable in explaining the variations observed between samples. it is based on the anova experimental design but analyzes the variance and determines the significance by permutations, as it is a non-parametric test [ ] . whereas, anova/manova assumes normal distributions and a euclidean distance, permanova can be used with any distance measure. the two analyses led to the same conclusions for this study. therefore their usefulness when used together as a tool to visualize and evaluate sample clustering was confirmed. the distinct clusters formed between the combination of pig farmers, and the air from pig buildings, and non-exposed individuals, is clearly linked to a strong divergence in the nasopharyngeal microbiota of farmers compared to other non-exposed individuals. mechanical deposition of < µm diameter inhaled particles on nasopharyngeal surfaces by inertial impaction [ ] , represents a continuous source of environmental bacteria to the nasopharynx. this continuous source of bacterial exposure may therefore be responsible for the establishment of a reservoir of bacteria reflecting long-term exposure (e.g., occupational exposure). a thorough understanding of the established bacterial community may then lead to a better evaluation of the risks associated with an environment. for example, domestic animals share some of their microbiota with their human cohabitants, supposedly through frequent and direct contact [ , ] . song et al., ( ) mention that airborne microbiota plays an important role in microbial transfer to the human upper respiratory tract. ten thousand litres of air are inhaled daily and the bioaerosols in the air may have an effect on the human nasal microbial community [ ] . finally, as different farm animals are associated with different microbiota, the normal nasopharyngeal flora of farmers may be differently disturbed. in other words, farmers working with different animals may have a different disturbances of their natural nasopharyngeal flora. therefore, the microbial fingerprint of the nasopharynx may be directly linked to a specific type of farming environment and the potential long-term health effects on farmers. the high abundance of firmicutes and bacteriotedes in pig buildings has been shown [ ] [ ] [ ] . interestingly, the results of this study are consistent with the literature, but with the added information indicating that, these same phyla colonize the nasopharynxes of farmers. more particularly, other studies have previously shown clostridia to be the most abundant class of bacteria in bioaerosols from pig buildings [ ] . this study not only confirmed the abundance of clostridia in air samples, but also that this class was the dominant class found in nasopharynx samples, while it was practically absent in non-exposed individuals. clostridium spp., identified as differentially abundant in the farmer nasopharyngeal samples in the present study, comprise potentially pathogenic species [ ] . specifically, clostridium butyricum, also differentially abundant in samples from farmers, has been identified as an emerging pathogen by public health authorities. some c. butyricum pathogenic strains were associated with the occurrence of necrotizing enterocolitis, a bowel disease [ ] . in addition, prevotella spp., which was strikingly more abundant in nasopharynx samples, is a well-known agent involved in upper respiratory tract infections [ ] [ ] [ ] . moraxella is another genus identified by the differential abundance analyses as being predominant in the nasopharynxes of farmers. like for other taxa identified in this study, strains of moraxella spp. were previously detected as airborne bacteria in pig buildings [ ] . the rate of colonization of moraxella spp. in healthy adult populations is around % [ ] . species of this genus are opportunistic pathogens responsible for upper and lower respiratory tract infections [ ] . taxa identified exclusively in nasopharyngeal samples and air samples from pig buildings could be candidates for new markers to assess exposure to bioaerosols in pig farming environments. although, actinobacteria are most abundant in the non-exposed controls, no pathogen was identified in the most abundant taxa presented in figure (except haemophilus influenza). an example of the most abundant actinobacteria in non-exposed control is micrococcus luteus that was differentially more abundant in controls compared to farmers. another example of firmicutes is staphylococcus epidermis that was more abundant in the controls compared to the farmers. the respiratory health of farmers has been of great interest for testing the hygiene hypothesis that stipulates that exposure to microbes from intensive farming during early life could be beneficial to health in adulthood [ , ] . however, the acceptance of the hygiene hypothesis is not unanimous in the scientific community [ ] . in this study, taxa identified as differentially abundant among farmers could hypothetically play a role in the prevention of allergy and the development of atopic diseases. indeed, some bacteria identified through this investigation (e.g., pedobacter, pelomonas, and megasphaera) have been linked to healthy respiratory conditions [ ] . a recent study, conducted by kraemer and colleagues, also found a distinct clustering between samples from the nasal cavities of pig farmers and air samples from their workplace, when compared to the nasal cavities of non-exposed individuals [ ] . moreover, samples from the nasal cavities of cow farmers clustered separately from pig worker samples and air samples from pig buildings [ ] . although the nasal cavity is a more transient environment for environmental bacteria than the nasopharynx, it confirms the hypothesis of a microbial fingerprint specific to the farming environment. it supports the idea of creating a worldwide database, that lists potential markers specific to certain environments, and to the nasopharynxes of the people working in them. this database could represent an important asset for associating bioaerosol exposure with health problems [ ] . the lack of a correlation between the nasopharynx of wastewater workers and bioaerosols from wwtps could be explained by the nature and duration of exposure. workers wear personal protective devices (e.g., masks) at certain working sites (e.g., biofiltration), which may affect the establishment of a 'new' environmental microflora. supporting this idea, the most abundant bacteria, shared by non-exposed controls and wastewater workers, are naturally occurring skin bacteria like propionibacterium, corynebacterium, staphylococcus, streptococcus, and cutibacterium. these taxa were not present (relative abundance less than %) in air samples from wwtps (data not shown). farmers do not usually wear respiratory protection and, moreover, they often live in the farming environment (e.g., in a house located near farms) and are consequently continuously exposed to the microbes generated from farming activities (occupational and residential exposures). therefore, the exposure that farmers are subjected to is more likely to modify their natural nasopharyngeal flora. the agricultural/non-agricultural hypothesis presented in this work regarding the nasopharyngeal flora of exposed workers should be validated in other agricultural and industrial environments. recent studies of the microbiome of the upper respiratory tract may have underestimated the influence of occupational exposure when considering the effect of a particular disease on the natural flora of upper respiratory tract tissue [ ] [ ] [ ] . the fact that the work environment may affect the natural flora of an exposed person on a long-term scale is a crucial consideration when he/she becomes a patient whose upper respiratory tract microbiota is the target of the disease. the results obtained in this work emphasize the importance of considering the environment of the nasopharyngeal flora of exposed workers, who are or could become patients suffering from chronic respiratory diseases. in the same way that recent advances in methods for identifying microbes has helped implicate the upper respiratory tract microbiome in inflammatory respiratory diseases, evaluating bioaerosol exposure can help us support the roles of resident microbes in both healthy and diseased tissues. specific human pathogens and antibiotic and zinc resistance genes were detected in the nasopharynxes of pig farm workers as well as in bioaerosols of pig buildings [ ] . for bacterial diversity analyses, the qpcr approach supports the use of the nasopharynx as an alternative to air sampling. the presence of zinc and antibiotic resistance genes, in the nasopharynxes of farmers, is explained by the use of zinc and antibiotics in animal farming (for therapeutic or sub-therapeutic use, such as for growth promotion) and implies possible human health risks. extensive use of zinc in pig feed is responsible for the proliferation of zinc-resistant bacterial communities at farms [ ] . cephalosporin is a commonly used antimicrobial drug in human infections and the spread of its resistance constitutes part of the antibiotic resistance crisis [ ] . finally, the mcr- gene was found in the nasopharynxes of half of the pig farmers in this study, although the use of colistin is extremely regulated in north america and limited to multi-drug resistant microbes [ ] . future studies should include detailed health information on the sampled individuals to investigate the nasopharynx microbiota associated with certain occupational health problems. additionally, a longitudinal study of the bacterial diversity, in the nasopharynxes of farmers and in bioaerosols from pig buildings, could unveil a long-term variation in microbial content. finally, information about the diet of exposed human (or animal) and antibiotic use could be added to the analyses as important factors influencing the microbiota. this is the first study to link the nasopharyngeal flora of exposed humans with the source of the exposure in an agricultural setting, using bacterial diversity analyses and the detection of specific pathogens and resistance genes. the results suggest that workers are carriers of bioaerosol-associated bacteria and that nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. furthermore, pig farmers are also carriers of specific human pathogens and resistance genes 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open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -o hmzaxm authors: kühnel, martina b; marchioro, linda; deffner, veronika; bausewein, claudia; seidl, hildegard; siebert, sarah; fegg, martin title: how short is too short? a randomised controlled trial evaluating short-term existential behavioural therapy for informal caregivers of palliative patients date: - - journal: palliat med doi: . / sha: doc_id: cord_uid: o hmzaxm background: informal caregivers of palliative patients show higher levels of depression and distress compared with the general population. fegg’s ( ) existential behavioural therapy was shortened to two individual -h sessions (short-term existential behavioural therapy). aim: testing the effectiveness of sebt on psychological symptoms of informal caregivers in comparison with active control. design: randomised controlled trial. setting/participants: informal caregivers of palliative in-patients. methods: the primary outcome was depression; secondary outcomes were anxiety, subjective distress and minor mental disorders, positive and negative affect, satisfaction with life, quality of life and direct health care costs. general linear mixed models allow several measurements per participant and change over time. reasons for declining the intervention were investigated by rosenstock’s health belief model. results: overall inclusion rate was . %. data of caregivers were available ( . % females; mean age: . years, standard deviation (sd): . ); participants were included in the main analysis. participation in sebt or active control was not significantly associated with post-treatment depression. outcomes showed prevailingly significant association with time of investigation. self-efficacy, scepticism of benefit of the intervention, belief of better coping alone and support by family and friends were significant factors in declining participation in the randomised controlled trial. conclusion: inclusion rate was tripled compared with a previously evaluated longer ebt group intervention. by shortening the intervention, inclusion rate was traded for effectiveness and the intervention could not impact caregivers’ psychological state. early integration of sebt and combination of individual and group setting and further study of the optimal length for caregiver interventions are suggested. • • informal caregivers of palliative patients are prone to higher levels of depression compared with the general population. informal caregivers are family members and other persons whose support of the patient is not financially rewarded. supporting informal caregivers is an essential part of palliative care, as defined by the world health organization. informal caregivers are prone to higher levels of depression, anxiety, strain and burden than the general population, , and the number of interventions to support them is growing. , however, a review of caregiver interventions identified a lack of proactive interventions and supposed that caregivers would prefer interventions that improve the ability to care. mindfulness-based interventions for caregivers could potentially close this gap. despite the challenges for caregivers to access interventions due to scheduling difficulties and them having to leave the patient alone, mindfulness showed positive influences on depression, strain and quality of life. fegg et al. developed existential behavioural therapy (ebt), an intervention aimed at informal caregivers of palliative patients. ebt was implemented in a group setting with a total of h focusing on mindfulness practice, strengthening resources, finding meaning, establishing self-care and developing personal values. medium to large effects on anxiety and quality of life and medium effects on depression were demonstrated. a weakness of this study was the low uptake of the intervention with . %. short-term existential behavioural therapy (sebt) aimed to be more compatible with caregivers' daily life. a qualitative study embedded in the fegg study had identified two ebt elements regarded as most helpful by caregivers: social support in the group and self-regulation via strengthening resources and practicing mindfulness. despite the support provided by the group, an individual setting was chosen for sebt to ensure a quicker start of the intervention. to condense ebt for the individual setting, sebt focused on the two elements of self-regulation, shortening it to two -h sessions. a feasibility study indicated that sebt was feasible and accepted by caregivers. although sebt is not a treatment applied for a disorder, the term 'therapy' was kept to mark the affiliation with ebt. this study's aim was to evaluate the effectiveness of the sebt intervention in comparison with a usual, nondirective psychological intervention using a randomised controlled trial study design. the primary outcome was informal caregivers' level of depression, as the fegg study had shown long-term effects on depression. secondary outcomes were informal caregivers' levels of anxiety, subjective distress and minor mental disorders, positive and negative affect, satisfaction with life, quality of life and direct health care costs. furthermore, we analysed caregivers' reasons to decline participation in the randomised trial as more research in this field had been suggested. this randomised controlled trial has a parallel-group design with equal : randomisation and four assessments: pre-treatment, post-treatment and follow-ups after weeks and months. we embedded a follow-up of those informal carers who declined to participate. the study was approved by the ethics committee of ludwig-maximilians-university of munich (no: - ) and was registered with clinical.trials.gov (nct ). informal caregivers were recruited from the munich university hospital palliative care unit, germany. inclusion criteria were minimum age of years and fluency in german. one caregiver per patient was included, preferably the person closest to the patient. excluded were professional legal representatives and caregivers with severe mental illness (e.g. dementia, acute addiction). the sample size was calculated according to fegg's study: psychotherapy research reports treatment effects between . and . standard deviation (sd). to achieve a power of . at % significance level using dupont what this paper adds? • • ebt was shortened to two individual -h sessions (sebt) to fit better into caregivers' daily lives. • • this randomised controlled trial tests the effectiveness of sebt on psychological symptoms of informal caregivers in comparison with an active control. implications for practice, theory or policy • • shortening the intervention tripled inclusion rate to . % reaching more caregivers. • • inclusion rate was traded for effectiveness and the intervention could not impact caregivers' psychological state. • • early integration of sebt and combination of individual and group setting are discussed. • • this study's results suggest further study of the optimal length for caregiver interventions. and plummer's sample size calculation and considering a dropout rate of %, participants were needed in every arm of the study. caregivers were approached earliest after the day of the patient's admission. they were screened for the inclusion criteria by psychologists with clinical experience. potential participants were contacted in person or by phone and informed orally and in written form about the study. caregivers who did not want to participate were asked to take part in the decliners' follow-up. all participating caregivers and patients provided written informed consent. consent of a legal guardian was sought for patients unable to give consent. immediately after making the first appointment and receiving the first questionnaire, participants were randomised by a randomisation list which was computergenerated with blocks of , each containing five control and five sebt assignments in random order. participants were informed about their allocation in the first session. the study was conducted on weekdays between january and february . recruitment was suspended for months due to staff change (march -august ) and three times due to staff vacation ( intervention sebt and control intervention both comprised two sessions in an individual setting lasting - min; appointments were arranged individually. the interventions took place in a separate room in the palliative care unit and in a psychotherapeutic practice. three psychologists with several years of experience in behavioural psychotherapy were trained using video feedback. sebt and control group sessions were audiotaped and rated for treatment integrity using coding guidelines and checklists (range - : ' ' = element missing to ' ' = fully consistent with the manual). control group. the active control group was oriented towards carl rogers' client-centred therapy, characterised by acceptance, congruence and empathic understanding, as recommended in supporting informal caregivers in a palliative setting. there was no mention of mindfulness or resources. sebt group. the first sebt session focussing on mindfulness included: introduction, psychoeducation about mindfulness, -min body scan, -min mindful breathing exercise, addressing questions and motivation to practice mindfulness every day using a cd provided. the second sebt session focussing on resources included: introduction, psychoeducation on psychological meaning of resources, encouragement to express strengthening areas and activities (based on schedule for meaning in life interview ), imaginative exercise of the inner image of the strongest resource addressing all five senses, choice of a symbol as reminder and prime, addressing questions, motivation to practice mindful breathing and imaginative exercise using the cd. caregivers' demographic data and patients' medical data were collected through self-report and clinic chart review. participants of the randomised controlled trial completed standardised questionnaires at the time of study entry (t ), after the second intervention session (t ) and weeks (t ) and months (t ) after the second intervention session. participants of the decliners' follow-up received three questionnaires: at t and follow-ups weeks (t ) and months (t ) after t , with no decliner questionnaire at t . all measurement instruments were used in a validated german version. primary outcome. level of depression was measured with patient health questionnaire, items; a score > is associated with clinical levels of depression, and scores are sums ranging from to . , secondary outcomes. generalised anxiety disorder was assessed using the generalised anxiety disorder questionnaire, items; a score > indicates general anxiety disorder, and scores are sums ranging from to . , subjective distress was measured using the national comprehensive cancer network's distress thermometer; a score > indicates a clinically relevant level of distress, scale range - , from 'no distress' to 'extreme distress'. minor mental disorders were assessed using the general health questionnaire, items, with higher scores indicating higher level of mental disorder; scores are sums of item values ranging from to . , positive and negative affect were measured using the positive and negative affect schedule, with higher scores indicating higher levels of affect; scores ranging from to are means of positive and negative items, respectively. , life satisfaction was measured using the satisfaction with life scale, with higher scores indicating higher degree of satisfaction; scores are sums of item values ranging from to . , quality of life was assessed using the world health organization quality of life questionnaire, abbreviated version: scores range from to , with higher scores denoting higher quality of life; scores were built according to the manual guidelines, including handling of missing data. , health-related resource use of the past months (number of physician contacts, physiotherapist contacts, hospital days, and rehabilitation days) was collected using the german questionnaire for health-related resource use in an elderly population (at t and t ). individual costs were added up after assigning a cost to each component based on unit prices published by bock et al. three numerical rating scales with one item each measured quality of life, physical impairment and psychological impairment, with scores ranging from to , with higher scores indicating higher levels. of all the used scales, only in the manual of the world health organization quality of life questionnaire, , guidelines on how to treat missing data were provided: outcomes were only computed if at least % of the items in a scale were available and the missing items were imputed with the mean of the available items. otherwise the whole observation was discarded. for consistency, we applied this approach to all scales. belief model, designed to predict health-promoting behaviour, was employed in order to understand reasons for declining. , the following four factors of the health belief model each comprised several variables and were included in questionnaires for decliners and for the randomised controlled trial. 'modifying factors': age, gender, knowledge about depression (numerical rating scale ranging from to ) and self-efficacy (german general self-efficacy short scale) scores are means ranging from to , with higher scores indicating higher levels. factor 'perceived susceptibility and severity': two numerical rating scales with - ranges on susceptibility for and severity of suffering from depression. factor 'perceived benefits and barriers': four numerical rating scales on scepticism of benefit of the intervention (adapted from patient questionnaire on therapy expectation and evaluation, - range), belief in benefit (adapted from german questionnaire for measurement of psychotherapy motivation, - range), belief one should cope alone (adapted from german questionnaire for psychotherapy motivation, - range), and belief that the intervention benefit would be greater than the costs ( - range). higher scores indicate higher agreement. factor 'cues to action': three numerical rating scales on advice from family/friends to accept psychological support, the extent of support by family/friends and the quality of the relationship with the patient, with higher scores indicating higher levels. changes in the outcomes over time were evaluated via general linear mixed model with random intercept for subjects. these models allowed several measurements per participant and change over time. a separate regression model was built for each outcome measure. outcomes from all three post-treatment questionnaires (t , t , t ) were dependent variables. variables 'group' (sebt or control group) and 'time of investigation' were independent variables. the interaction effect between 'group' and 'time of investigation' was only included if significantly different from zero. the pre-treatment (t ) value of each outcome measure was included as a predictor variable, capturing individual status before the treatment. in all models, we controlled for age, gender, relationship with the patient (patient is partner/child vs other); patient's time of death (patient alive, unknown, deceased > months before measurement, < months before measurement); employment (employed/student vs retired/unemployed); the psychologist delivering the intervention (psychologist , or ); and other support used (e.g. social worker, pastoral care, other psychologist; yes, no or unknown). besides the main model (model ), we conducted sensitivity analyses considering the following two subgroups of the study population: only participants (sebt or control group) who attended both interventional sessions (model ) and all control participants and only sebt participants who had practised mindfulness at least once using the cd (model ). sensitivity analyses controlling for missing data were also conducted. data were analysed according to the principle 'full analysis set' which is as complete and as close as possible to the intention to treat ideal of including all randomised subjects. the regression analyses included only individuals with at least one intervention session and participation in the investigations before (t ) and after the intervention (t ). a binary logistic regression was conducted to investigate which factors led to declining or accepting the intervention. based on rosenstock's health belief model, stepwise inclusion of four factors emulated the process of decision-making for or against the intervention. an overall result was deduced from all four steps. in addition, linear mixed models with repeated measurements were used to model all outcome parameters at t , t and t in order to detect differences in outcomes between the participants of the randomised controlled trial and the decliner participants. to analyse differences in direct health care costs, the non-parametric mann-whitney u test was used due to skewed distribution of the data. statistical analyses were performed using ibm spss statistics v. ; a value of p < . was considered significant; a value of p < . was considered a trend. results are reported following the consort statement. out of potential participants, were excluded during recruitment ( . %; see figure ), hence caregivers were contacted ( . %). of these, participated in the decliners' follow-up and declined any participation. a total of caregivers were randomised into the sebt or the control group; the inclusion rate was . %. during the study, cases were excluded as they had been wrongfully assigned. after the randomisation, participants dropped out before t ( sebt, controls). in total, participants of the randomised controlled trial took part in the pre-intervention examination (t ). at t , sebt and control participants showed no significantly different characteristics (see table ). the mean age was . years (sd . ) and most participants were female ( . %). more than one-third of the participants held a university degree ( . %), more than half were married ( . %); nearly one-third was retired ( . %) and twothirds employed (full time . %, part time . %). participants were mostly either patients' partners (including wives or husbands; . %) or their children ( . %). cancer was the prevailing diagnosis of the patients ( . %). two-thirds of participants received interventions by psychologist ( . %). most patients were alive at t ( . %; . % deceased ⩽ months ago; . % unknown; n = ). at t , patients were mostly alive ( . %) or had deceased during the last months ( . %; . % unknown; n = ). at t , most patients had deceased during the last months ( . %; . % alive; . % deceased > months ago; . % unknown; n = ), and at t , most patients had a due to missing data at t , participants' datasets were excluded from analyses. b despite missing data at t , participants' datasets were included in analyses. thirty participants of the pre-intervention examination (t ) were not included in the main data analysis because they dropped out during the intervention or had missing data at t (see figure ). an independent-sample t-test indicated that these drop-outs had higher levels of negative affect at t (mean: . , sd: . ) than participants included in the main analysis (mean: . , sd: . ; p-value: . ), and they tend to higher levels of minor mental disorders (mean: . , sd: . ) than participants included in the main analysis (mean: . , sd: . ; p-value: . ; unequal variances). they did not significantly differ in any other outcome or characteristic. a total of participants were included in the main regression analysis (model ) according to the principle of 'full analysis set' as they participated in at least the first two investigations at t and t . these participants showed mild and subclinical levels of depression at t (mean: . , sd: . ). the sample's average score on anxiety was just under the cut-off for clinically relevant levels (mean: . , sd: . ). their average level of distress was above the score indicating clinical relevance (mean: . , sd: . ). at t , datasets were available and included in analyses. at t , datasets were available and included. the percentage of scales with at least one missing item was . %. by including observations which had at least % of items completed, we were able to lower the number of scales that had to be discarded to . %. in total, intervention sessions were held (sebt and control, including dropouts), participants received only one session. audiotapes of the intervention sessions were available ( . %), eight were incomplete and not rated; five participants declined consent for audiotaping. audiotapes were rated to evaluate treatment integrity. the therapists' adherence to the intervention manual was high (sebt mean: . , sd: . ; control mean: . , sd: . ). the level of depression did not differ significantly between sebt and control group (sebt beta: -. ; control group as reference category); this was true for all three models (see table ). apart from the impact of pre-treatment depression, there was a trend for the time of investigation being associated with the post-treatment depression level (t beta: -. ; t beta: - . ; t as reference category), as depression was on average lower at t and t than at t . the interaction effect between the group and the time of investigation was not included in the main model since it was not significantly different from zero. according to the results of the main models, all posttreatment secondary outcomes did not significantly differ between sebt and control group (for tables see supplemental material appendix b). the interaction effect between the group (sebt/control) and the time of investigation was not included in the main models as it was not significantly different from zero, except for psychological impairment. time of investigation was significantly associated with outcomes anxiety (t beta: - . ; t beta: - . ; t as reference category), positive affect (t beta: . ; t beta: . ) and minor mental disorders (t beta: . . ; t beta: . ), and was associated by trend with negative affect (t beta: . ; t beta: . ) and quality of life (numerical rating scale; t beta: . ; t beta: . ). patients' time of death was significantly associated with outcomes negative affect (alive beta: . ; deceased > months ago beta: -. ; time of death unknown beta: . ; deceased ⩽ months ago as reference category), satisfaction with life (alive beta: - . ; deceased > months ago beta: . ; time of death unknown beta: - . ), subjective distress (alive beta: . ; deceased > months ago beta: - . ; time of death unknown beta: . ) and psychological impairment (alive beta: . ; deceased > months ago beta: . ; time of death unknown beta: . ); patients' time of death showed a trend to be associated with anxiety (alive beta: . ; deceased > months ago beta: - . ; time of death unknown beta: . ). relationship with the patient was significantly associated with quality of life (numerical rating scale; partner/child beta: -. ; other relationship as reference category). age showed a trend to be associated with subjective distress (beta: . ) and gender showed a trend to be associated with psychological impairment (male beta: -. female gender as reference category). in addition, we conducted sensitivity analyses regarding missing data (for tables see supplemental material appendix c). participants without missing items in any of the relevant outcome scales were regarded as having no missing data (n = , . %); they were significantly younger than participants with missing data (n = ). a variable discriminating between these two groups was added to an additional set of regression analyses. these analyses yielded highly similar results compared the analyses described above, apart from the variable missing data being associated by trend with negative affect (no missing data beta: . ). at t , a mann-whitney u test showed that, at t , there was no significant difference between the median of direct health care costs for the past months of sebt participants (median: € , n = , interquartile range: . ) and controls (median: € , n = , interquartile range: . ). at t , there was also no significant difference between the median of direct health care costs for the previous months of sebt participants (median: € , n = , interquartile range: . ) and controls (median: € , n = , interquartile range: . ). data of decliners were available at t , as dropped out before t . data of decliners were available for the follow-up at t and data of decliners at t . declining participants were significantly older (mean: . years, sd: . ) than participants of the randomised controlled trial (mean: . , sd: . ) but did not significantly differ regarding gender, relationship status or employment. linear mixed models with repeated measurements modelling all outcome parameters at t , t and t showed no differences in any outcomes between the participants of the randomised controlled trial and the decliner participants. the binary logistic regression showed that the preference towards the decliner study significantly depends on 'perceived benefit and barriers' and 'cues to action' (see table ). the odds to prefer the decliners' follow-up were . times higher for caregivers with high self-efficacy ( % confidence interval: . - . ), . times higher when being sceptic of the benefit of the intervention ( % confidence interval: . - . ), . times higher for caregivers who believed in better coping alone ( % confidence interval: . - . ) and . times higher for caregivers supported by family and friends ( % confidence interval . - . ). the purpose of sebt is to provide a short-term intervention with coping strategies to informal caregivers of palliative patients facing the existential situation of disease and bereavement. this randomised controlled trial studied the impact of sebt on depression, anxiety, subjective distress, minor mental disorders, positive and negative affect, satisfaction with life, quality of life and direct health care costs. receiving sebt sessions or supportive psychological sessions was neither significantly associated with the primary outcome of post-treatment depression nor with the secondary outcomes. the outcomes were prevailingly associated with their respective level before the intervention and with the time of investigation, which leads to the assumption that the time passing was the main reason for changes of outcomes over the course of months. caregivers who declined the intervention did not differ significantly from participants of the randomised controlled trial in outcomes at any assessment. in fegg et al'.s randomised controlled trial on ebt, the control participants did not receive a control treatment and instead could decline any support or could choose from the spectrum of available support at the palliative care unit (e.g. physicians, nurses, chaplains, social workers, psychologists and bereavement group), whereas this study included an active control group. it is possible that sebt and control showed no significant difference as both groups received a treatment of similar effectiveness. palliative caregivers' capacities for learning new skills like mindfulness might be limited: they face high emotional distress and the responsibilities palliative caregivers typically take over for the patient (i.e. financial decisions, organisation of follow-up hospice care) additionally to their own duties. fegg et al'.s study provided a group setting which could have facilitated learning the new skill of mindfulness by benefitting from group cohesion, central for beneficial effects in group therapy , or by relieving participants from the personal responsibility to practice. participants in sebt were asked to practice mindfulness by themselves which was possibly too demanding, leading to low compliance to practice and less effectiveness. our aim was to create a short-term ebt intervention that fitted better into informal caregivers' daily lives. we reached our goal of increasing acceptability: . % of all contacted caregivers participated in the randomised controlled trial. shortening ebt and choosing an individual setting tripled the inclusion rate compared with . % in fegg et al'.s study. however, by shortening the intervention, we traded inclusion rate for effectiveness and the intervention was not intensive enough to impact caregivers' psychological state in comparison to the control group. carmody and baer's review about the optimal length of mindfulness based programmes, with participants ranging from healthy to chronically ill participants, did not evidence that shortened versions of mindfulness-based programmes are less effective compared with the standard format of class hours. the authors suggested that adaptations including less class time may be worthwhile for populations for whom a longer time commitment may be a barrier to participate. but how short is too short? the study with the fewest sessions in carmody and baer's review included -weekly -h classes, which is three times more instruction time than in this study. our results lead to the conclusion that the -h sebt version is too short, especially with participants as burdened as palliative caregivers. the optimal length of mindfulness-based interventions for informal caregivers should be investigated further to offer interventions which impact caregivers' psychological status while not overwhelming them. strengths of the study include the randomised controlled design, the high adherence of the therapists to the manual and the embedded decliners' follow-ups which allowed a comparison with trial participants and ensured high external validity. during the study, it became apparent that participants had been assigned to sebt or control group violating the randomisation protocol. recruiting was suspended, all data collected up to this point was carefully checked and affected participants' data were excluded from analysis. in addition, appropriate measures of staff change and staff training were taken. data of caregivers were removed from analysis as they had missing post-intervention data at t or dropped out of the intervention. comparing their pre-intervention data to the other participants, they had higher levels of negative affect and of minor mental disorders which possibly caused them to drop out. this leads to the assumption that the intervention might be too demanding for highly burdened caregivers. profiting of the 'small window' for recruiting caregivers before they become too burdened by care could be facilitated with early integration of palliative care. , early integration of sebt could help caregivers learn new skills to prepare for stressful times ahead. furthermore, sebt could benefit from mixing the settings. sörensen et al. suggested combining group and individual setting to improve caregiver affect in the individual setting and help build social networks in the group. individual sebt could offer immediate support to caregivers, while a following ebt group could yield higher impact on caregivers' psychological morbidity with more class hours and positive influence of group cohesion on motivation and personal practice. ethical approval precludes the data being provided to researchers who have not signed the appropriate confidentiality agreement. these restrictions are as per the ethics committee of ludwig-maximilians university munich which approved the study . in accordance with ethical approval, all results are in aggregated form to maintain confidentiality and privacy. data are held at the department of palliative medicine, munich university hospital, ludwig-maximilians-university, munich, germany. the author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: stifterverband für die deutsche wissenschaft e.v. 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into standard oncology care: asco clinical practice guideline update summary how effective are interventions with caregivers? an updated meta-analysis we thank all caregivers, patients and staff members especially verena zierl, marianne schmidt and sigrid haarmann-doetkotte for their contribution to this study. key: cord- - hnlu v authors: sutthiruk, nantanit; botti, mari; considine, julie; driscoll, andrea; hutchinson, ana; malathum, kumthorn; cucunawangsih, cucunawangsih; wiwing, veronica; puspitasari, vivien; shanmugakani, rathina kumar; akeda, yukihiro; kodera, takuya; santanirand, pitak; tomono, kazunori; yamanaka, takayuki; moriuchi, hiroyuki; kitajima, hiroyuki; horikoshi, yuho; lavrinenko, alyona; azizov, ilya; tabriz, nurlan; kozhamuratov, margulan; serbo, yekatherine; yang, dahae; lee, woonhyoung; bae, il kwon; lee, jae hyun; lee, hyukmin; kim, jung ok; jeong, seok hoon; lee, kyungwon; peremalo, thiba; madhavan, priya; hamzah, sharina; than, leslie; wong, eng hwa; desa, mohd nasir mohd; ng, kee peng; geronimo, marionne; tayzon, maria fe; maño, maria jesusa; chow, angela; hon, pei-yun; win, mar-kyaw; ang, brenda; leo, yee-sin; chow, angela; hon, pei-yun; see, tina; ang, brenda; marin, rocio alvarez; de sousa, marta aires; kieffer, nicolas; nordmann, patrice; poirel, laurent; laochareonsuk, wison; petyu, sireekul; wanasitchaiwat, pawin; thana, sutasinee; bunyaphongphan, chollathip; boonsomsuk, woranan; maneepongpermpoon, pakpoom; jamulitrat, silom; sureshkumar, dorairajan; supraja, kalyanaraman; sharmila, soundararajan; cucunawangsih, cucunawangsih; setiawan, benny; lumbuun, nicolaski; nakayama, haruo; ota, toshiko; shirane, naoko; matuoka, chikako; kodama, kentaro; ohtsuka, masanobu; bacolcol, silverose ann andales; velmonte, melecia; alde, allan; chavez, keithleen; esteban, arlene joy; lee, aisa jensen; hsieh, tai-chin; shio-shinjean; huang, huey-jen; huang, shu-ju; huang, yu-huan; cheng, pei-chen; yu, su-fang; tsao, shih-ming; lee, yuan-ti; li, chien-feng; lu, min-chi; pruetpongpun, nattapol; khawcharoenporn, thana; damronglerd, pansachee; suwantarat, nuntra; apisarnthanarak, anucha; rutjanawech, sasinuch; cushinotto, lisa; mcbride, patty; williams, harding; liu, hans; hang, phan thi; anh, dinh pham phuong; le, ngai; khu, dung; nguyen, lam; castillo, roel beltran; sureshkumar, dorairajan; gopalakrishnan, ram; ramasubramanian, venkatasubramanian; sreevidya, subramanian; jayapradha, ranganathan; umetsu, atsushi; noda, tetsuhiro; hashimoto, kenyuu; hayashi, akihiro; kabashima, mikie; jadczak, ursula; elvelund, knut; johnsen, marit; borgen, bente; lingaas, egil; mao, chia-hua; chang, fu-chieh; liu, chang-pan; chao, ru-hui; chang, fu-chieh; liu, chang-pan; pawapotako, junpen; prasertpan, chadanan; malaihuan, wantanee; uirungroj, phisit; prasertpan, chadanan; saenjum, chalermpong; ouirungrog, teerapat; uirungroj, phisit; borrell, sue; bass, pauline; worth, leon; xian-li, zhao; xiao-long, li; xue-hua, yao; wei, ren; zeng, zhang xia; kong, man ying; lai, christopher koon chi; lee, suet yi; tsang, ngai chong; o’donoghue, m. m.; boost, m. v.; suen, l. k. p.; siu, g. k.; mui, k. w.; lai, c. k. c.; tsang, d. n. c.; sato, yuka; tateishi, mariko; mihashi, mutsuko; flor, jose paulo; bautista, marko; de roxas, v. jay; vergara, justine; añonuevo, nicolo andrei; kwek, marion; acuin, jose; sanchez, anna josea; bathan, avel; jantan, jamilah binte; guek, chua chor; kian, eu chiow; pirido, pampe anak; aron, nur fadilah binte mohd; estacio, leah may; palana, francis alvarez; gracia, michelle; shamsuddin, nur syafiqah binte; castro, kersten timbad; baloria, madonna; adam, faezah binte; wei, zhang; fong, poh bee; kalisvar, marimuthu; chow, angela; ang, brenda; chuang, i-ju; yi-chuncho; chiu, yu-fen; chen, lung-chih; lin, yi-chun; dong, shao-xing; lee, yi-chieh; kuan, hui-chen; lin, hsin-hua; chi, chia-chun; lu, chin-te; chang, fu-chieh; liu, chang-pan; ya-fen, tang; li-hsiang, su; jien-wei, liu; chao, hsuehlan; changchien, pinru; chen, weifang; lai, chunghsu; ara, lutfe; mowla, syed mohammad niaz; vashkar, shaikh mahmud kamal; chan, wai fong; chunyau, mabel yin; lingchong, karen kam; onli, tze; kaur, rajwinder; yan, ng po; chiu, gloria chor shan; cheung, christina w. y.; ching, patricia t. y.; ching, radley h. c.; lam, conita h. s.; kan, c. h.; lee, shirley s. y.; chen, c. p.; chan, regina f. y.; leung, annie f. y.; wong, isadora l. c.; lam, s. s.; chan, queenie w. l.; chan, cecilia; kaur, rajwinder; nematian, seyed sadeq seyed; palenik, charles john; askarian, mehrdad; nematian, seyed sadeq seyed; palenik, charles john; hatam, nahid; askarian, mehrdad; nakamura, itaru; fujita, hiroaki; tsukimori, ayaka; kobayashi, takehito; sato, akihiro; fukushima, shinji; matsumoto, tetsuya; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; vergara, justine; de roxas, v. james; kwek, marion; flor, jose paulo; bautista, marko; vergara, justine; de roxas, v james; andreiañonuevo, nicolo; kwek, marion; ho, yeng may; kum, jia qi; poh, bee fong; marimuthu, kalisvar; ang, brenda; liu, tzu-yin; chu, sin-man; chen, hui-zhu; chen, tun-chieh; chen, yichun; tsao, ya-ching; skuntaniyom, sumawadee; malathum, kumthorn; tipluy, pirawadee; paengta, sangwan; wongsaen, ratchanee; thanomphan, sutthiphun; tariyo, samettanet; thongchuea, buachan; khamfu, pattama; thanomphan, sutthiphan; songtaweesin, wipaporn natalie; anugulruengkit, suvaporn; samransamruajkit, rujipat; sosothikul, darintr; tansrijitdee, ornanong; nakphunsung, anry; srimuan, patchareeyawan; sophonphan, jirachaya; thanyaweeputhanakit; payuk, kunyanut; picheansathian, wilawan; viseskul, nongkran; denardo, elizabeth; leslie, rachel; cartner, todd; barbosa, luciana; werner, heinz-peter; brill, florian h. h.; kawagoe, julia yaeko; de nardo, elizabeth; wilson, sarah edmonds-; macinga, david; mays-suko, patricia; duley, collette; hang, phan thi; hang, tran thi thuy; hanh, tran thi my; gordon, christopher; sureshkumar, dorairajan; durairaj, roopa; rohit, anusha; saravanakumar, saujanya; hemalatha, jothymani; hirano, ryuichi; sakamoto, yuichi; yamamoto, shoji; tachibana, naoki; miura, miho; hieda, fumiyo; sakai, yoshiro; watanabe, hiroshi; velmonte, melecia; bacolcol, silverose ann; alde, allan; chavez, keitleen; esteban, arlene joy; lee, aisa jensen; chow, angela; lim, jia-wei; hon, pei-yun; hein, aung-aung; tin, grace; lim, vanessa; ang, brenda; chow, angela; hein, aung-aung; lim, jia-wei; hon, pei-yun; lim, vanessa; tin, grace; ang, brenda; chow, angela; tin, grace; hein, aung-aung; lim, vanessa; lim, jia-wei; hon, pei-yun; ang, brenda; chao, huwi-chun; yeh, chiu-yin; lo, mei-feng; chao, huwi-chun; piwpong, chonlada; rajborirug, songyos; preechawetchakul, ploypailin; pruekrattananapa, yada; sangsuwan, tharntip; jamulitrat, silom; wongsaen, ratchanee; paengta, sungwan; nilchon, napatnun; thanompan, sutthipun; tariyo, samattanet; le, ngai; khu, dung; kolesnichenko, svetlana; azizov, ilya; lavrinenko, alyona; tishkambayev, yerbol; lavrinenko, alyona; azizov, ilya; tishkambayev, yerbol; alibecov, asylkhan; kolesnichenko, svetlana; serbo, yekaterina; nam, youngwon; park, jae hyeon; hong, yun ji; kim, taek soo; park, jeong su; park, kyoung un; kim, eui-chong; aziegbemhin, samuel abumhere; enabulele, onaiwu; tung, yao-shen; chen, an-chi; huang, shen-min; yang, yui-yein; wu, li-hung; lin, chin-cheng; chang, fu-chieh; liu, chang-pan; lien, tzu hao; chang, jia hao; huang, yu shan; chen, yi shun; saenjum, chalermpong; sirilun, sasithorn; ouirungrog, teerapat; ouirungroj, phisit; trakulsomboon, suwanna; prasajak, patcharee; kwok, maryanne w. n.; ng, lady s. h.; wong, lindy m. t.; poon, lenina s. l.; lai, mary k. l.; cheng, holly h. s.; fong, s. k.; leung, cindy f. y.; hasegawa, jumpei; shirakawa, hiroki; wakai, sachiko; mieno, makiko; hatakeyama, shuji; tateishi, mariko; mihashi, mutsuko; sato, yuka; saenjum, chalermpong; deeudom, manu; tharavichitkul, prasit; ouirungrog, teerapat; ouirungroj, phisit; chinniah, terrence; tan, jackson; prabu, kavitha; alam, sartaj; wynn, aung kyaw; ahmad, rashidah; sidek, amalina; samsuddin, dg azizah; ajis, noraini; ahmad, aliyah; magon, susylawathi; chu, boon; kuang, jiqiu; gao, yan; wang, shoujun; hao, yunxiao; liu, rong; li, dongmei; wang, hui; yan, ng po; nishio, hisanori; mori, hitomi; morokuma, yoshiko; yamada, takaaki; kiyosuke, makiko; yasunaga, sachie; toyoda, kazuhiro; shimono, nobuyuki; babenko, dmitriy; turmuhambetova, anar; cheşcă, antonella; toleman, mark a.; babenko, dmitriy; turmuhambetova, anar; cheşcă, antonella; toleman, mark a.; babenko, dmitriy; turmuhambetova, anar; azizov, ilya; cheşcă, antonella; toleman, mark a.; akhmaltdinova, lyudmila l.; turmuhambetova, anar; cheşcă, antonella; babenko, dmitriy; magsakay, mark albert; macatibag, angelo; tayzon, maria fe; lerios, jeannica kriselle; azizov, ilya; lavrineko, alyona; babenko, dmitry; sheck, eugene; edelstein, mikhail; liu, tzu-yin; li, lih-yue; chan, chiung-wen; pan, hui-chuan; chen, tun-chieh; vanishakije, wipa; jaikampun, warisra; cheng, pei-chen; huang, huey-jen; huang, shu-ju; huang, yu-huan; li, su-yin; yu, su-fang; li, jian-feng; wu, yu-ping; lee, yuan-ti; lin, chiao-hui; chang, ping-chin; tariyo, samatanet; paengta, sangwan; wongsaen, ratchanee; thanompan, suttsiphan; skuntaniyom, sumawadee; malathum, kumthorn; sukkra, suchada; zaman, khalequ; zaman, sheikh farzana; zaman, farzana; aziz, asma; faisal, sayeed-bin; traskine, magali; ruiz-guiñazú, javier; borys, dorota; zaman, khalequ; zaman, sheikh farzana; zaman, farzana; aziz, asma; faisal, sayeed-bin; traskine, magali; ruiz-guiñazú, javier; borys, dorota; lam, wendy wai yee; chow, may; choy, lucy; kam, joseph; salleh, sharifah azura; yacob, razila; yusof, siti rokiah; jalil, nordiah awang; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; de roxas, v. jay; vergara, justine; millan, maria lourdes; kwek, marion; acuin, jose lito; lee, aisa jensen; velmonte, melecia a.; bacolcol, silverose ann a.; alde, allan; chavez, keitleen; esteban, arlene joy; ting, ching-i; dissayasriroj, sunisa; chinniah, terrence rohan; prabu, kavitha; ahmad, rashidah; magon, susylawathi; dinisuhaimi, jauharatud; mirasin, aizzuddin; morni, nurul; chu, boon; samsuddin, azizah; ahmad, aliyah; sidek, amalina; ajis, noraini; abubakar, amalina; shafiee, amanie; safar, julaini; yan, ng po; annie, leung; ling, fung yuk; edna, lau; kristine, luk; shinomiya, satoshi; yamamoto, kumiko; kjiwara, kayoko; yamaguchi, mitsuhiro; chow, angela; tin, grace; zhang, wei; hon, pei-yun; poh, bee-fong; marimuthu, kalisvar; ang, brenda; chan, ming-chin; wang, chih-chien; huang, shu-ju; huang, huey-jen; yu, su-fang; huang, huan-yu; cheng, pei-chen; li, jian-feng; lee, yuan-ti; lai, chiung-ling; lu, min-chi; kosol, sajeerat; sakolwirat, wantana; paepong, patchanee; jansanga, sawalee; jaisamoot, pattarin; thongnuanual, nuttha; srithong, chittima; somsakul, somporn; malathum, kumthorn; plongpunth, sutima; punpop, mukkapon; malathum, porntip; malathum, kumthorn; thanomphan, sutthiphan; wongsaen, ratchanee; peautiwat, kulada; boon kirdram, nattawipa; picheansathian, wilawan; klunklin, pimpaporn; samethadka, geetha; suzuki, naoko; asada, hitomi; katayama, masao; komano, atsushi; sato, akihiro; nakamura, itaru; watanabe, hidehiro; matsumoto, tetsuya; seo, hye kyung; hwang, joo-hee; shin, myoung jin; kim, su young; kim, eu suk; song, kyoung-ho; kim, hong bin; un, lai-si; vong, choi-ian; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; de roxas, v. james; vergara, justine; kwek, marion; koh, jocelyn; agustinus, sherly; hassan, rozita bte abu; thinn, yin phyu; ng, benjamin; tun, soe pyae; ha, su mon thi; xiaoting, xue; li, lin; chuang, leyland; niroshika, attanayaka mudiyanselage chulani; perera, kaluarachchige anoma kaluarachchi; fernando, dimingo kankanamalage diana grace; hemamala, bodhipakshage rohini; yeh, chiu-yin; chao, huwi-chun; yang, hui-chun; chiu, hsiang-ju; shih, ya-ling; chien, yu-shan; lin, wan-yi; pan, chia-yun; chang, ying-yun; yea, chiu-yuch; chu, ming-hsien; lee, li-chu; chiu, hsiang-ju; shih, ya-ling; yang, hui-chun; yu-hsiu, lin; siao-pei, guo; pak-on, leung; mei-fe, sie; jyh-jou, chen; yu-hsiu, lin; yong-yuan, chang; kuo, shu-yuan; lin, yu-hsiu; zhang, ji-sheng; leung, pak-on; sie, mei-fe; chen, jyh-jou; chen, yan-ru; lin, yu-hsiu; chen, ying-ling; taou, chi-fen; chen, hsiao-shan; tang, hung-jen; chen, shin yu; chen, yin yin; der wang, fu; shih, tzu-ping; chen, chin-yu; chen, su-jung; wu, mei-chi; yang, wan-ju; chou, mei-ling; yu, man-ling; li, li-chu; chu, cheng-wei; tsou, wen-hao; wu, wen-chih; cheng, wen-chi; sun, cho-ching; shih, tzu-ping; chen, chin-yu; lu, shu-hua; chen, su-jung; yang, hsin-ling; lu, cheng-yu; yu, man-ling; li, li-chu; chu, cheng-wei; tsou, wen-hao; wu, wen-chih; cheng, wen-chi; sun, cho-ching; hirunprapakorn, nitchawan; malathum, kumthorn; apivanich, sirilux; pornmee, ttipakorn; beowsomboon, chonnikarnt; rajborirug, songyos; pruekrattananapa, yada; sangsuwan, tharntip; jamulitrat, silom; kumkoom, itthaporn; kasatpibal, nongyao; chitreecheur, jittaporn; kasatpibal, nongyao; whitney, joanne d.; saokaew, surasak; kengkla, kirati; heitkemper, margaret m.; apisarnthanarak, anucha; muntajit, thanomvong; apivanich, siriluk; malathum, kumthorn; somsakul, somporn; phan, hang thi; dinh, anh pham phuong; nguyen, tuyet thi kim title: abstracts from the th international congress of the asia pacific society of infection control (apsic): bangkok, thailand. - february date: - - journal: antimicrob resist infect control doi: . /s - - - sha: doc_id: cord_uid: hnlu v nan antimicrobial resistance (amr) is a major problem worldwide. antimicrobial stewardship (ams) has the vital aim of ensuring optimal use of antimicrobial medicines to minimize amr. new strategies are needed to reduce amr. it is vital to ensure that key stakeholders are involved in the development of these strategies. this study aimed to examine key stakeholders' perspectives on the underlying causes of amr in thailand. semi-structured interviews were conducted with key multidisciplinary clinicians, heads of department and healthcare administrators who were involved in ams programs in a , -bed university hospital in bangkok thailand. qualitative content analysis was used to analyze the interview data. one of the key themes that emerged was lack of regulatory control resulting in widespread antibiotic availability and use both in health and agriculture in thailand, including over-the-counter availability of antibiotics. this ease of accessibility combined with poor consumer knowledge was considered one of the most important contributors to the increasing prevalence of amr. the development and implementation of more effective infection prevention and control strategies was identified as a priority, particularly in healthcare. three major concerns related to the perception that many patients admitted to hospital already have amr infections, that staff prescribing behaviors are not ideal, and that the lack of resources to develop and implement ams programs is an important barrier to decreasing the overuse of antibiotics. participants recognized that amr is a major problem in thailand and in healthcare. there was agreement that what is required is better regulatory control of antibiotics and medical engagement in ams. background antimicrobial resistance is a major problem of post-operative neurosurgical infection over the recent years. this study aimed to evaluate an increasing trend of infection in neurosurgical patients and susceptibility pattern of the causative pathogen. material and methods over a period of five years (june to june ), cerebrospinal fluid and pus samples derived from clinically suspected cases of post-operative neurosurgical infection were processed using the standard procedures for culture and antibiotic susceptibility testing. of these patients, causative pathogens were identified in patients ( . %). majority of infections were caused by multidrugresistant gram-negative bacilli (mdrgnb) including pseudomonas aeruginosa (n = , . %), acinetobacter baumannii (n = , . %), sphingomonas paucimobilis (n = , . %), escherichia coli(n = , . %), aeromonas salmonocida (n = , . %), and klebsiella pneumoniae (n = , . %). the common isolates showed a high susceptibility to tigecycline ( . %) and amikacin ( %), ceftriaxone ( . %) and ceftazidime ( %). all gram-positive bacteria isolates were susceptible to tigecycline and vancomycin. although multidrug-resistant (mdr) gram-negative bacilli (gnb) become a global concern, the disease burden of mdr gnb in children has not been reported yet in japan. we elucidate the impact of invasive mdr gnb infections among japanese children in the hospital setting. materials and methods a primary questionnaire was sent to pediatric training facilities. a secondary questionnaire was sent to determine whether any cases showed a positive blood or cerebral spinal fluid culture for extended spectrum beta-lactamase (esbl) producing gnb, ampc β-lactamases producing gnb, or carbapenem-resistant enterobacteriacae (cre) between april and march .the following data were collected; demographic data pertaining to both the care facilities and patients, clinical diagnosis, and outcomes. the response rate for the primary questionnaire was %. among facilities that responded, facilities were eligible for the secondary questionnaire. the response rate for secondary questionnaire was %. a total of pediatric patients had invasive mdr gnb infection. the median age was . years old (interquartile range months- years old). the number of patients with bacteremia caused by esbl gnb, ampc gnb, and cre were ( %), ( %), and ( %), respectively. the clinical diagnosis of esbl and ampc gnb showed cases of sepsis. the clinical diagnosis of cre showed cases of catheter related blood stream infection and cases of sepsis. mortality at days for esbl, ampc and cre bacteremia was %, % and %, respectively. the most common mdr gnb bacteremia was esbl gnb among children in this survey. tuberculosis is often complicated by the addition of non-specific inflammation, which changes not only the clinical manifestation of tuberculosis, but the course and outcome of disease. this study aimed to study the spectrum of non-specific microflora from patients with active tuberculosis and to evaluate its susceptibility to antimicrobial agents. the study was conducted in - ; sputum samples were investigated. identification of microorganisms was carried out by maldi-tof methods using mass-spectrometer microflex (bruker daltonics, germany). the sensitivity of microorganisms to antibiotics was determined by disk-diffusion methods (clsi ). statistical processing and data analysis was performed using whonet . program. the growth of non-specific microflora in patients with tuberculosis was obtained in % of cases. the predominant etiologic role in non-specific inflammation belonged to s. aureus ( %), k. pneumoniae were isolated in . %, a. baumanniiin . %. remaining microorganisms were isolated in individual cases. . % staphylococci were mrsa, to other anti-staphylococcal drugs s. aureus has kept a high sensitivity. isolated k. pneumoniae strains were resistant to cephalosporins of the rd generation in . %, the resistance to meropenem marked in . %. a. baumannii was characterized by a high resistance to antibiotics - . % esbl-producing strains, . % and . % strains were resistant to imipenem and meropenem, % a. baumannii strains were resistant to fluoroquinolones. conclusions according to antibioticogram data, the isolated microorganisms obtained from non-specific microflora of patients with tuberculosis, may adversely affect the course of the disease and impede the selection of antibacterial drugs and affect the outcome of the disease. rising number of candidiasis significantly contribute towards resistance of commonly used antifungal agents. lately, candida species such as c. rugosa and c. pararugosa have emerged as fungal pathogens that cause invasive infections. material and methods clinical isolates were from two tertiary referral hospitals in malaysia. test for antifungal susceptibility, biofilm, protease and phospholipase activities, all of which contribute to their virulence were performed. biofilms were quantified using crystal violet (cv) and tetrazolium (xtt) reduction assays in -well microtiter plates. time point reading was done on all strains incubated at , , , and hours. there were seven isolates of c. rugosa and one isolate of c. pararugosa in this study. e-test antifungal tests showed that all candida rugosa strains were susceptible-dose dependent towards voriconazole and resistant to fluconazole, amphotericin b and caspofungin based on clinical and laboratory standard institute guidelines. highest biomass was observed in one of the c. rugosa strains, followed by c. pararugosa at hours of incubation. however, highest bioactivity was observed in the atcc at hours, followed by c. pararugosa at hours and the same c. rugosa train at hours. virulence was also contributed by secretion of protease enzymes by all the clinical strains. none of the c. rugosa and c. pararugosa trains showed any phospholipase activity. conclusions c. rugosa and c. pararugosa clinical isolates should be considered pathogenic species because of their resistance against commonly used antifungal drugs and their contributing virulence factors. in response to an antimicrobial resistance "apocalypse" the medical city, a private tertiary hospital in the philippines, conducts microbiologic surveillance and has an existing "prior approval of restricted antibiotics" wherein release of identified broad spectrum antibiotics were done only upon approval of id consultants. in june , a second tier of asp was introduced. the "drug duration, audit and feedback" (ddaf) program monitors and audits the duration of empiric antibiotics prescribed by clinicians. sticker reminders are being placed on the chart on day as a reminder to de-escalate and on day as a reminder to consider stopping the antibiotics. this study aimed to present a comparative study of the antimicrobial resistance of the top bacteriologic agents from respiratory isolates in a tertiary care hospital in the philippines, from january to june versus january to june , as a surrogate marker to the success of the second tier of asp recently introduced materials and methods most prevalent organisms from sputum, endotracheal aspirate and bronchoalveolar lavage were determined through laboratory surveillance comparing their resistance pattern from january to june versus january to june . the top respiratory pathogens in the icu were identified. some decrease in the resistance data of the most common isolate, klebsiella pneumoniae were as follows: % decrease in resistance to ceftriaxone, % to levofloxacin and % to piperacillin-tazobactam. similar decreases in resistance were seen with pseudomonas aeruginosa and other organisms. the study showed decrease in resistance of most common bacteriologic agents from respiratory isolates upon introduction of ddaf program. methicillin-resistant staphylococcus aureus (mrsa) is a growing clinical problem in subacute wards where patients have a longer length of stay than in acute wards. universal antiseptic baths could be added to the armamentarium for mrsa prevention and control. our study aimed to assess for the baseline antiseptic susceptibilities in mrsa, prior to the institution of universal antiseptic baths in subacute wards. we conducted a cross-sectional study, testing for susceptibilities to chlorhexidine and octenidine in mrsa isolates obtained from inpatients of two subacute wards from may-july . minimum inhibitory concentrations (mics) of chlorhexidine and octenidine were determined by the modified clinical and laboratory standards institute (clsi) methods, with microbroth dilution susceptibility testing for a range of . - ug/ml. results a total of mrsa isolates were tested: in may, in june, and in july. for chlorhexidine, all except for one had mic = . the remaining with mic = had occurred in july . in comparison, the majority ( . %) of the isolates had mic = . when tested for octenidine susceptibility, with the remaining having an mic = . a higher proportion of mrsa isolates with the higher mic level (mic = ) to octenidine was observed in june than in the other months, although statistical significance was not achieved due to the small sample size (or . , %ci . - . , p = . ). conclusions mrsa isolated from patients from subacute wards were highly susceptible to chlorhexidine and octenidine. universal antiseptic baths could be implemented in subacute wards, with follow-up studies conducted to monitor for any development of antiseptic resistance. antimicrobial activity of octenidine against multidrug-resistant gram-negative pathogens rocio alvarez marin , , marta aires de sousa , nicolas kieffer , patrice nordmann , , laurent poirel multidrug-resistant gram-negative (mrgn) pathogens pose a major and growing threat for health care systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. well tolerated antiseptic molecules may be a very useful implementation in infection control,not only to reduce the dissemination of methicillin-resistant staphylococcus aureus (mrsa), but also mrgn. as decolonization strategies with regard to mrsa are already implemented in high risk areas (i.e. icus), this study aimed to investigate, if the same protocol might be concomitantly efficient against mrgn. a series of different species (escherichia coli, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, pseudomonas aeruginosa) was studied to prove efficacy under clinically relevant conditions according to an official test norm (en ). we used clonally-unrelated isolates per species, including a single wild-type strain, and four mrgn isolates, corresponding either to the mgrn or mgrn definition of multidrug resistance. octenidine (oct, schuelke&mayr gmbh, germany) susceptibility was evaluated with and without organic load. results a contact time of seconds or minute was fully effective for all isolates by using different oct concentrations ( . % and . %), with a bacterial reduction factor of > log systematically observed. growth kinetics were determined with two different wild-type strains (a. baumannii and k. pneumoniae), proving a time-dependent efficacy of oct, mirroring what has been previously observed for mrsa. these results highlight that oct, besides being a very effective agent against mrsa, may also be extremely useful to eradicate emerging highly resistant gram-negative pathogens associated with nosocomial infections. acinetobacter baumannii is an important opportunistic nosocomial pathogen causing a variety of infections. the intrinsic virulence of drug-resistant a. baumannii has remained controversial. we compared mortality rates and sepsis score of patients with a. baumannii bacteremia caused by different level of drug resistance. materials and methods a retrospective study was conducted in adult patients (age > years) admitted to songklanagarind hospital during and and blood culture positive for a. baumannii after days of admission. antimicrobial resistance was categorized into four levels comprising of non-multidrug resistance (non-mdr), multidrug-resistant (mdr), extensively drug-resistant (xdr), and possible pandrug-resistant (possible-pdr). severity of underlying disease of the patients immediately before onset of bacteremia was determined by sequential organ failure (sofa) score and american association of anesthesia (asa) score. virulence of a. baumannii was assessed in terms of sepsis score and in hospital mortality rate. the study identified , , , and cases of bacteremia caused by non-mdr, mdr, xdr, and possible pdr, respectively. after adjusting for confounding effect by using cox proportional hazard model, mortality rates attributable to a. baumannii was significantly associated to levels of drug resistance. using non-mdr as a reference, the incidence rate ratios and corresponding % confidence intervals ( % c.i) of mdr, xdr, and possible pdr were . ( % c.i = . - . ), . ( % c.i = . - . ), and . ( % c.i = . - . ) respectively. the virulence of a. baumannii did not loss with drug resistance. most of the antibiotic stewardship programs (asp) in the developing world measure antibiotic consumption, adherence to antibiotic guidelines and antibiotic resistance. however, antibiotic associated diarrhea (aad) is a common medical problem of antibiotic treatment and important quality monitor of asp was not monitored commonly in india. this study aimed to measure the prevalence of aad in hospitalized patients receiving antibiotics. materials and methods a point prevalence study was conducted in a -bed tertiary care cardiac hospital in chennai, south india. all hospitalized patients in cardiology wards and intensive care units (icus) receiving at least one dose of either oral or intravenous antibiotic were audited by physician assistant for the symptoms of diarrhea and cross checked by interviewing patients. during the study period, eligible patients had available records for analysis. there were patients ( . %) receiving antibiotics. of these, there were receiving single antibiotic, receiving two antibiotic combinations, receiving three antibiotic combinations and were taking four antibiotic combinations. the details of diarrhea was missing in patients' medical records. only patients ( . %) developed diarrhea and they received two antibiotics combination. conclusions although more than half of the patients received antibiotics, aad was not common in our hospital. however, regular monitoring of aad along with other parameters were required for better implementation of asp in the hospital. antimicrobial resistance and infection control , (suppl ):as background infections caused by antibiotic-resistant bacteria have led to increase burden on the healthcare system. effective antimicrobial stewardship control program (ascp) requires the clinician acceptance of program recommendation. we evaluate the antibiotic consumption and antibiotic susceptibility after ascp implementation in in a teaching hospital in tangerang, indonesia. our ascp restrict the prescription of carbapenems, fourth generation cephalosporins, and tigecycline. antibioticsusceptibility and consumption of restricted antibiotics were extracted from database. antibiotics use was measured by the number of ddds per bed-days results the proportion of susceptible bacteria against; cefpirome increased from % to %, cefepime % to %, imipenem % to %, and tigecycline % to % during to . the proportion of meropenem susceptibility remained the same at % in and . the defined daily doses (ddds) per bed-days was significantly reduced in all restricted antibiotics from to except tigecycline. the consumption of . g cefepime was , and , . g cefpirome was , and , . g meropenem was , and , . g teicoplanin was . , . and , . g vancomycin was , and . , and . g linezolid was . , and in , and , respectively conclusions our ascp was effective in terms of lowering broad spectrum antibiotic consumption and improving the antibiotic susceptibility. utilization of diagnosis-procedure combination data for advancing the antimicrobial stewardship program haruo nakayama, toshiko ota, naoko shirane, chikako matuoka, kentaro kodama, masanobu ohtsuka toho university ohashi medical center, tokyo, japan background infection with antibiotic-resistant bacteria results in increased morbidity, mortality and economic burden. antimicrobial stewardship program (asp) has been widely implemented to guide appropriate antibiotic use, in order to minimize antibiotic resistance. however, establishment of asp is not always possible due to lack of interest. we examined the application of diagnosis-procedure combination (dpc) data as an incentive for achieving the target of asp. the toho university ohashi medical center inpatient initiated asp focusing on reduction of inappropriate perioperative antibiotics and anti-mrsa drugs. the dpc data was extracted for antibiotic consumption and duration in each patient from april to march . the consumption of the first-generation and second-generation cephalosporins as perioperative antimicrobial agents was % during observation period. this proportion was below the initial benchmark. on the other hand, the consumption of anti-mrsa agents was . % higher than the benchmark. more than half of patients undergoing surgery received perioperative antibiotics only one day. the majority was cardiovascular surgery patients used intraoperatively. our study shows that utilization of the dpc database for advancing the asp is possible. it is convenient process to measure outcome of asp at the group or organizational level. background extensively drug-resistant acinetobacter calcoaceticus-baumannii complex (xdr-abc) pneumonia is an important cause of healthcareassociated pneumonia. although tigecycline was not approved for treatment of healthcare-associated pneumonia, it has been used offlabel for xdr-abc pneumonia. we evaluated whether the clinical efficacy of tigecycline combined with aerosolized colistin methanesulfonate (cms) is superior to aerosolized cms alone. this is a retrospective case-control study, conducted in wan-fang medical center, taipei medical university, taipei, taiwan from november to february . the definition of xdr-abc pneumonia was pneumonia caused by abc with susceptibility only to colistin and tigecycline. cases were patients who received aerosolized cms in combination with intravenous tigecycline for at least days to treat xdr-abc pneumonia. controls were those who received inhaled cms alone and were selected based on the following matching criteria to cases; age (± years), acute physiology and chronic health evaluation (apache) ii score (± points). there were patients in each group. the mean age of patients was years old. the proportion of patients underwent mechanical ventilation were . % and . % in cases and controls, respectively (p = . ). the median apache ii score was . ( . - . ) in cases and . ( . - . ) in control group (p = . ). the mean length of hospital stay was days (p = . ), -days mortality rate was % and . %(p = . ), and overall mortality was . % and . % (p = . )in cases and controls, respectively. conclusions despite the active in-vitro susceptibility of tigecycline against xdr-abc, combination therapy with tigecycline and aerosolized cms for xdr-abc pneumonia showed no additional clinical benefit. the effectiveness of reducing the amount of antibiotic resistant strains by promoting antibiotic stewardship program of a medical center huey-jen huang , shu-ju huang ,yu-huan huang ,pei-chen cheng , su-fang yu , shih-ming tsao , , yuan-ti lee , ,chien-feng li , , min-chi lu in , the amount of antibiotics in this medical center accounted for . % of its total drug amount. the calculated did dosage of inpatient antibiotics was and the rate of antibiotic-resistant strains has been on the rise. to cope with the development of drug resistance, from to , the antibiotic stewardship program (asp) was executed to promote proper use and therefore to decrease the volume of antibiotics, and to reinforce mdro isolation. materials and methods a multi-discipline team for antibiotic stewardship was reformed. id doctors and infection control practitioners developed regulations for antibiotic use, conducted training programs, reviewed antibiotic uses and gave feedback. the compliance and accuracy of hand hygiene, and isolation precaution and protection were strengthened by nursing personnel. the pharmacist team division provided daily antibiotic assessment and statistics. microbiology laboratory was responsible for drug-resistant data. the expenditure of consumed antibiotic, as a ratio of total drug, declined from . % in to . % in . from to , the did from to , carbapenems from . to . , quinolones from . to . , and glycopeptides from . to . . furthermore, crpa from . % to . %, crab from . % to . %, mrsa from . % to . %, vre from % to %. however, a little elevation of crkp from . % to . % was observed. employing asp, we have enhanced the cooperation among antibiotic team members. as a result, the correct use of antibiotics was improved, the amount of antibiotics was less consumed, and the ratios of most mdro declined. antimicrobial resistance and infection control , (suppl ):as background increased antibiotic resistance among escherichia coli has led to inappropriate empirical antibiotic use (iau) for associated infections. limited data exists for iau among cases with acute uncomplicated cystitis (auc). we conducted a prospective observational study at a general practice (gp) clinic from december to february . eligible participants included women aged - years with auc. all participants' urine cultures were sent before empirical antibiotics were prescribed at the gp physicians' discretion. the rate of iau was subsequently identified by the investigators. strategies to minimize iau were then determined based on the relevant data of auc treatment in this study. eighty participants were enrolled. e. coli was the most common pathogen isolated ( . %) with resistance rates to trimethoprimsulfamethoxazole, fluoroquinolone, ceftriaxone, amoxicillin-clavulanate and ertapenemof . %, . %, . %, . % and %, respectively. extended-beta-lactamase production was confirmed in . % of e. coli isolates. the rate of iau was . %. ciprofloxacin use was the only independent risk factor for iau (adjusted odds ratio, . ; . - . ; p = . ). based on the study results, including the in-vitro susceptibility data and the risk factors for acquisition of antibiotic-resistant e.coli, a specific algorithm for auc treatment was created. if this algorithm was used along with education about iau and antibiotic stewardship program focusing on ciprofloxacin use, the rate of iau would have decreased to . %. our findings suggest the high rate of iau in auc treatment in a gp setting and underlie the need for multifaceted interventions to reduce iau. background infection control/prevention (ic) programs seek to prevent infections via surveillance and outbreak investigation, hand hygiene, isolation precautions, environmental disinfection, evaluating ic products, and policy development. antimicrobial stewardship programs (asp) try to optimize antibiotic use for better outcomes and less toxicity. both reduce antimicrobial resistance in pathogens, educate healthcare staff, patients, and families, and optimize resource use. our study objectives were to define areas of synergy between ic and asp efforts with specific examples and to determine how best to promote these. bryn mawr hospital: -bed community-teaching hospital with ic practitioners; asp team has one infectious diseases (id) physician and an id-trained pharm.d. it is part of a hospital system with microbiology laboratory. asp has been in placed for - / years. synergy between asp and ic: ( ) collaborative identification of outbreaks (e.g., regional babesiosis in , ongoing c. difficile cases), ( ) monitoring antimicrobial resistance via complementary computer surveillance systems, ( ) coordinating presentation of microbiology antibiograms, ( ) collaboration on healthcare staff educational programs, and ( ) joint presentations to system wide committees and accrediting organizations. from to dosage days for selected antibiotics decreased - % per patient-days and total antibiotic cost decreased $ , ( %) per year. conclusions ic and asp programs should work together to the benefit of both and the institution and health system as a whole. this can be facilitated by regular communications and meetings, ongoing review of microbiological pathogen and susceptibility trends, and collaboration on research and educational programs. healthcare workers (hcws) play an important role to be a consultant about antibiotic use for patients. this would be helpful to reduce antibiotic overuse and prevent emergence of antimicrobial resistance in the hospital and public settings. we conducted across sectional study by using a self-assessment validated questionnaires, to determine current awareness and common habits related to antibiotic usage and antimicrobial resistance among hcws in hung vuong hospital; an obstetrics and gynecology hospital in vietnam. a total of hcws were enrolled in the survey. although % of hcws responded correctly "many infections are becoming increasingly resistant to treatment by antibiotics", % of them thought that "antibiotic resistance occurs when your body becomes resistant to antibiotics". a total of % ( % ci: . - . ) and % ( % ci: . - . ) of hcws have correct knowledge about antibiotic usage and antimicrobial resistance. a total of % of hcws answered that they have to prescribe antibiotic because they cannot follow up the patients' condition. twenty-two percent of hcws answered that it is necessary to take antibiotic when people have fever. antimicrobial resistance and infection control , (suppl ):ds the global community demands standardization to approaches in patient safety. protocols differ to meet the specific demands of health care facilities; patient safety has always been the common end. an introduction and understanding of compliance under the australian setting would reaffirm this common end. our similarities and differences in achieving the purpose are interesting to note.this study aimed to share information between infection control professionals, how australia kept pace with the technological evolution of reusable medical devices (rmds) and caters to specific reprocessing requirements to ensure patient safety. how rmds are reprocessed to minimize, control and prevent healthcare-associated infections (hais) materials and methods the australian commission on safety and quality in healthcare requires health service organizations to comply with standards ensuring patients get the quality of care they truly deserve. all cssd throughout australia are responsible for: standard . antimicrobial resistance and infection control , (suppl ):ds background fumigation of operating rooms (or) with high concentration of toxic chemicals is an age old tradition practiced in most of the developing world to control hospital acquired infections. this approach lost favor in the developed world due to questionable efficacy and toxicity concerns. however, most of the hospitals in developing world continue to use fumigation practices with variable frequency. here we report our experience of fumigation free or in india. this quasi-experimental before and after intervention study was conducted in a -bed tertiary care referral women and children hospital in chennai (south india) between january and september . the practice of or disinfection using quaternary ammonium compounds fumigation was allowed in addition to standard cleaning methods in before-intervention phase (jan to dec ). in after-intervention phase (jan ) onwards the fumigation practice was stopped and standard cleaning methods alone followed. the monthly environmental microbiological surveillance cultures and surgical site infection (ssi) rates were compared and analyzed. in the before-intervention phase there were surgical procedures were carried out with ssis and environmental samples tested all were within acceptable limits as per defined standards. in the after-intervention phase surgeries were carried out with no ssis and all environmental samples collected were within acceptable limits. the standard cleaning methods alone without chemical fumigation is sufficient for operating rooms disinfection in india. however, this finding should be confirmed in large multi-site studies before universal recommendation. antimicrobial resistance and infection control , (suppl ):ds background it is reported that skin antiseptic with a chlorhexidine-alcohol concentration of more than . % usedduring blood cultures lowers contamination rate more effectively than povidone-iodine skin antiseptic. as an approach to enhance precision of blood cultures, i heldcampaigns for appropriate sterilization methods in also changed % povidone-iodine to % chlorhexidinealcohol antiseptic during blood cultures in , and reported results. material and methods study periods:april, -march, (a), august, -july, after the sterilization methods campaign(b), and august, -july, after the antiseptic change(c). i investigated and compared each period. the contamination was calculated by dividing the number of cases in which there was only one positive result of two sets or more of blood culture specimens submitted on the same day by the total of all paired sets collected. the contaminants were defined as cons, bacillus spp., corynebacterium spp., micrococcus spp., propinonibacterium spp. which a doctor took as causative organism of the infection were excluded. the contamination of the blood culture for periods a: . %, b: . %, and c: . %. there's significant decrease in the contamination during blood cultures after sterilization methods campaign (a-b)(p < . ). it suggests contamination decreased by performing appropriate sterilization methods. there were no contamination differences in b-c after changing disinfectant (p = . ). it was suggested that an equal skin sterilization effect was provided when i performed the sterilization with % chlorhexidine-alcohol and % povidone-iodine, which was appropriate.the % chlorhexidine-alcohol has an immediate effect and durability in comparison with povidone-iodine, shorting the time for drawing blood after sterilization. reflecting importance for busy on-site blood cultures. antimicrobial resistance and infection control , (suppl ):ds background lovisenberg diakonale hospital has olympus edt machines for cleaning of endoscopes. in the standard program the wash time is minutes with special detergent and the disinfection time is minutes. periodical testing of final rinse-water is performed regularly with good results. microbiological control of the final rinse-water from the washer-disinfector for endoscopes (ewd) is the most widely used methods for detecting growth of bacteria. the validation process is a comprehensive procedure that requires both resources, time, knowledge, special equipment and access to the microbiological laboratory. the hospital wanted to assure the quality of decontamination of their endoscopes. we have established a partnership with the department of infection control at oslo university hospital in order to validate the ewd. the decontamination of flexible endoscopes must be tested and validated according to the standard en iso . to validate the disinfection process and secure a repeatable method, we used a surrogate endoscope (spypach). this is equipped with biological indicators, temperature sensors and pressure-and flowmeasures. microbiological control of final rinse-water: satisfactory results according to standard.cultivation of biological indicators: unsatisfactory results according to standard. discovery of protein and fibrin in surrogate endoscope: unsatisfactory results according to standard. measurement of temperature, pressure and flow: no deviation conclusions the endoscope was not adequately cleaned during decontamination. final rinse-water had satisfactory quality but remaining biological indicators and protein residues showed that the decontamination was not satisfactory. alternative solutions may include: increasing the wash time and/ or modify the contents of detergent. antimicrobial resistance and infection control , (suppl ):ds background as we know, the biological indicators are the most accepted means of the qc in sterilization. because the biological indicators contain geobacillus stearothermophilus that have spore inside the cell, if the sterilization were successful, the g.sterothermophilus should be killed, and the biological indicator can't detected by color or machine. so, in this study, we aimed to make sure whether the super rapid biological indicator can replace the traditional biological indicator or not. according to the instruction of the super biological indicator, after minutes, the data will show pass or not. so we using the culture method to make sure the result are the same or not. in this study, we collected super rapid readout biological indicators from feb. to mar. , and all the result showed that was no bacteria growth after hour incubated. then we used thioglycolate and tsa to make sure the results . according to the experiment's result, all of the culture data showed that super rapid readout biological indicators were the same. the accuracy were % and the sensitivity and specificity were % and %. antimicrobial resistance and infection control , (suppl ):ds background sterilization wrap is commonly used for instrument trays or cassettes. there are many different types and sizes of wraps available. typically, two sheets are needed to provide an effective barrier and a specific technique is recommended [cdc, aami st ] to allow for aseptic opening. wrapped instruments should be secured with sterilization tape that also serves as an external indicator. before closing, a multiparameter chemical indicator should be included inside along with the instruments. before this study, we used cotton of wraps. and the problem we focused was the expiry date. according to the taiwan cdc suggestion, the cotton's expiry date is days. but it's too short for us. this study aimed to find some types that can replace the cotton. in this study, we used the crepe paper of wraps to replace cotton. otherwise, we want to elongate the expiry date. so we collected the instruments that cover by crepe paper and that storage in csr (hepa level: ) for , , , , , , , weeks. then we used the broth methods to make sure whether the instruments were contamination or not. in this study, we found all of the instruments were clean on weeks. after weeks, the culture result showed that some bacteria were growth on the instruments. according to the experiment's results, we suggested that the crepe paper can replace the cotton of wraps, and it could elongate the expiry date for weeks in our csr. antimicrobial resistance and infection control , (suppl ):ds in , pieces of the mouth mirrors from dental unit were broken as they were packed and mixed with the other tools that were heavy, shape, and without protection. it broke the mouth mirrors from a process of packaging. the important is to prevent the broke of mouth mirror in order to have the good quality and enough instruments to service patients.this study aimed to determine the result of development of sterilization process toprevent the broke of mouth mirror in cssd of wanonniwas hospital. the sample group was composed of ) mouth mirror and ) personnel from the cssd persons.the process included steps: ) prepare process is to learn the reason of the breaking of mouth mirror and create the system in every process from caring, washing, packaging, sterilization, keeping, sending to dental unit, ) methods process is inform the personnel about the reason, why mouth mirror were broken, change the methods of sterilization of mouth mirror, and ) evaluation : this study were collected during fiscal year through . the breaking of mouth mirror in year , , and (may) were pieces, , and pieces, respectively. it has a clearly decrease. the sterilization to prevent the broke of mouth mirror that caring keep the mouth mirror into the box with a lid. the process of washing and drying, sorting the mouth mirror. packing cloth wrapped. sterilization, distribution and storage kit for the side impact protection. antimicrobial resistance and infection control , (suppl ):ds the medical instrument cleaning and disinfection procedures are important process to remove organic and inorganic matter, due to concerns about contamination risks especially protein residue, to prevent cross-contamination and ensure the safety. this study aimed to determine the cleaning efficacy of two different cleaning methods, manual cleaning process and an automatic washer disinfector machine, using the protein residue check test in the central sterile supply department (cssd), maharaj nakorn chiang mai hospital. the , , and samples of medical instrument washed and cleaned by manual cleaning process, the automatic washer disinfector machine, and re-sterile (without washing process), respectively, were collected from july to august, to determine the protein residue. the protein residue was detected with two different detections, the protein residue check test (pose health care co., ltd., thailand) and fluorescence-based protein detection test (lab focus co., ltd.). the ( . %), ( . %), and ( . %) samples of manual cleaning process, the automatic washer disinfector machine, and re-sterile produced undetectable protein residues, respectively (p-value = . ). the ( . %), ( . %), and ( . %) samples of manual cleaning process, the automatic washer disinfector machine, and re-sterile produced detectable protein residues, respectively. the results indicated that, the medical instrument using the re-sterile process should pre-clean instrument with enzymatic detergent to remove gross soil immediately and must be thoroughly washed and cleaned before being sterile. moreover, the manual cleaning process and an automatic washer disinfector machine must be optimized to eliminate protein contamination and minimize the cross-contamination. background different medical device cleaning and disinfection procedures are used on a large scale. it is an important procedure to remove organic and inorganic from medical device to prevent cross-contamination. this study aimed to determine the cleaning efficacy of an alkaline detergent used in an automatic washer disinfector machine in the central sterile supply department (cssd), maharaj nakorn chiang mai hospital. an alkaline detergent was developed by pose health care co., ltd., thailand. the programs were designed with different concentrations of alkaline detergent , , , and ml ( . , . , . , and . v/v, respectively) and two different temperatures ( and °c). the cleaning efficacy was monitored using a tosi (en iso ) and brown stf loaded check strips. each program used three tosi test kits and five brown stf strips to evaluate the cleaning efficacy. additionally, protein residue was detected with the pyromol® test, protein residue check test, and fluorescence-based protein detection test. moreover, biofilm and microorganisms were determined using a scanning electron microscope (sem). the optimum concentration of the alkaline-based cleaning detergent and temperature were ml ( . v/v) and °c, respectively. this program ( ml and °c) produced undetectable protein residues, biofilm, and microorganisms on medical instruments after the cleaning process. comparable to the recent condition of ml ( . v/v) concentration and at °c, the optimum condition of ml concentration and at °c reduced the cleaning costs by % per each cleaning process. background carbapenemase-producing enterobacteriaceae (cpe) infections are an emerging threat in specific regions of south east asia, europe, and the united states. cpe occurs sporadically in australia with isolated genomic clusters. different classes of carbapenemases are prevalent by region worldwide, and in australia, imipenemase-producing cpe (bla imp- ) is endemic at low levels. in our healthcare facility, carbapenemases other than imp demonstrate epidemiological links to recent overseas healthcare or residency. following retrospective recognition of a cluster of cpe cases linked to a single healthcare facility in melbourne, the local health authority, the victorian department of health and human services, released comprehensive consensus guidelines for the management of cpe in december . based on a "search and destroy" strategy, instruction is provided regarding identification of cpe, centralised reporting, contact tracing and screening, the need for alerts, clearance protocols for contacts, cleaning validation and -monthly point-prevalence surveys. oversight of outbreaks is provided by a state incident management team and a single state reference laboratory performs genomic analysis of all isolates. this presentation will outline the experience and lessons learned in implementing these cpe guidelines in our healthcare facility. examples include the development of new systems to identify and communicate cpe cases, electronic alerts for isolation and screening, a cpe staff education program and methods for conducting pointprevalence surveys in high-risk wards. early identification and screening of patients hospitalised abroad and the need for a functional it platform to facilitate electronic flagging/ alerts represent challenges likely to be faced by many healthcare facilities in our region. in recent years, we found rising in trends of measles infections, even in a small local epidemic. it may because of the measles virus' genes per se, antigenic variation or other factors. infected with measles virus can cause temporarily decline in human's immune, especially cellular immunity. lack of effective immune reactions can lead to secondary bacterial and multiple infections. we investigated measles-infected patients and evaluated susceptible factors for multiple drug-resistant bacteria infections. we also examined hospital infection prevention and control protocol in controlling measles outbreaks in our hospital. during to july , cases of patients with measles were detected in our hospital. we retrospectively reviewed and analyzed measlesinfected cases who had multiple drug-resistant bacteria infection data. we found that the main risk factors to be infected with multiple drug-resistant bacteria included: ) age under months, ) abnormal cardiac functions, ) having malnutrition and ) having encephalitis. the hospital should focus on patients under months of age, with abnormal cardiac functions, having malnutrition or encephalitis to avoid multiple drug resistant bacteria infections and decrease mortality by strengthening treatment and implementation of infection prevention and control protocols. the global spread of carbapenemase-producing enterobacteriaceae (cpe) is a major challenge for infection control practitioners.we adopted a proactive approach that all cpe carriers were isolated in a designated ward with strict contact precautions. here, we investigated if cpe can survive terminal disinfection in hospital environment. our study aimed to evaluate the extent of cpe contamination in patient care environment after terminal disinfection using microbiological sampling. microbiological samples for cpe were taken from the general wards' environment whenever a patient newly identified with cpe was removed for isolation. environmental samplings were collected by trained personnel. high-touch and wet surfaces were sampled using sterile polywipe sponge. chromid carba agar was used for selective cultivation of cpe. suspicious colonies grown after overnight incubation at °c were further examined for carbapenemase production using carba-np. we confirmed carbapenemase production using genexpert carba. between april and october , environmental samples were collected from wards. we found . %tested positive for cpe that included hand-wash basins, one sink and one hospital curtain. these isolates were imp-producing cpe. six affected basins/ sinks were cleared from cpe after cleaned with detergent followed by disinfection with . % sodium hypochlorite solution ( , ppm) daily for one week. the cpe in the remaining two hand-wash basins survived for days after daily decontamination and the basins need to be replaced. our results highlighted hand-wash basins may serve as a potential environmental reservoir for cpe. as standardized decontamination regimen for sinks was lacking, we recommended hand-wash basins should not be used for the disposal of body fluids. terminal cleaning of isolation rooms is an essential step in infection control. however, traditional cleaning and disinfection may be inadequately performed by time limitation. ultra-violet (uv) devices are effective in environmental decontamination but their cost can be prohibitive. recently, a more economical version of a uv-c sterilizing unit has become available but its effectiveness for environmental decontamination has not yet been independently evaluated. our study aimed to evaluate the effectiveness of the spectra uvc light system to reduce viability of healthcare-associated pathogens in a ward environment. four organisms (staphylococcus aureus, enterococcus faecalis, acinetobacter baumannii and klebsiella pneumoniae) were coated onto designated areas of formica that were then attached to eight locations in the room. the room was irradiated for minutes and the lamp was moved to a second position then treatment was repeated. cultures were performed and resulting colonies were enumerated. surviving numbers were compared with non-irradiated controls. all organisms were rendered non-viable in areas receiving direct irradiation or substantial reflected light. at two sites where heavily shaded (rear of bedside lockers and armchairs), a -log reduction in viability of e. faecalis was observed. conclusions spectra uvc light offered an effective adjunct to conventional terminal disinfection of isolation rooms. although two irradiation periods using two positions of the lamp were needed, to ensure that shaded areas would receive adequate treatment, the total time for disinfection was only minutes. as uv treatment does not produce residues, there is no down time after use. the current status of cross-infection risks in hotels yuka sato, mariko tateishi, mutsuko mihashi kurume university, kurume, fukuoka, japan in japan, the ministry of health, labour, and welfare published the "guidelines for the prevention of new strains of influenza infections in employers and employees [ ] ". however, our survey, which investigated infection control for new strains of influenza in , revealed that recognition of these guidelines was low, showing that infection prevention control had been insufficiently implemented in hotels. our study aimed to identify the current status of cross-infection risks in hotels, and use the results to improvethe control of infectious diseases in such areas. the study ran from march - , . the study volunteers were members of the all japan ryokan hotel association. to assess environmental contamination levels in hotel settings, the atp and amp swab test kits (kikkoman lumitester pd- ) were used. frequently touched surfaces were measured for contamination. measurement sites included that were measured intermittently, and that were measured before and after cleaning. there were intermittently measured monitoring sites that exceeded , rlu including the open/close buttons inside the kitchen elevator and first floor handrails. there were monitoring sites measured before and after cleaning that exceeded , rlu even after cleaning, including the inner sides of restaurants' sliding doors and the inner washroom door knobs of guest rooms. conclusions atp values of more than , rlu were detected at some monitoring sites, suggesting the need to reconsider methods and frequency of cleaning by taking risk of cross-infection into account. the purpose of this study was to test the effectively of the infection control risk assessment (icra) monitoring tool developed by the infection prevention and control unit (ipcu) of asian hospital and medical center with the aim to increase the compliance of construction workers to recommended infection prevention and control measures during construction, renovation and demolition in the hospital. materials and methods indicated in the icra monitoring tool were the details of the activity and the infection risk level (class i,ii,iii and iv). the design used wasa quasi-experimental designwhich was conducted among all construction projects in the hospital within a -year period. the percent compliance was computed by number of compliant projects per month over total number of monthly projects which thenmultiplied by . there were a total of construction projects monitored by direct observation which utilized the icra tool. other interventions included orientation of construction workers to the tool, acknowledgment and accountability of recommended infection prevention and control measures by signing the tool and lastly, making use of the tool to provide feedback. results show an improvement in the compliance to infection prevention and control interventions from average of % during pre-intervention to % post intervention. having an icra tool paved the way for construction workers to be pro-active and be involved in preventing infections brought by construction, renovation and demolition. national kidney foundation, singapore, singapore; jurong west dialysis centre, singapore, singapore; kolam ayer dialysis centre, singapore, singapore; woodlands dialysis centre, singapore, singapore; teck whye dialysis centre, singapore, singapore; yishun dialysis centre, singapore, singapore; hougang dialysis centre, singapore, singapore; kim keat dialysis centre, singapore, singapore; tampines dialysis centre, singapore, singapore correspondence: jamilah binte jantan (jamilah.jantan@nkfs.org) antimicrobial resistance and infection control , (suppl ):e in the dialysis centre, there is potential for cross transmission of infectious agents through contaminated devices, hands, equipment, supplies and environmental surface during haemodialysis (hd) treatment. to reduce the risk of acquiring infections, staff routinely clean and disinfect medical equipment and high-touch areas after each patient's hd treatment at the national kidney foundation (nkf). this study aim of the study is to assess environmental cleaning of hightouch areas and develop intervention program to achieve compliance ≥ %. materials and methods this is a quantitative study involving infection control link nurses (iclns) at the community-based dialysis centres (cb-dcs), nkf from october to april . in november , iclns conducted an environmental cleaning assessment of high-touch areas using a checklist and glo germ kits, to ascertain the efficacy of environmental cleaning at cb-dcs. pre-study data showed an overall average of % compliance. rca revealed the absence of an audit tool for high-touch areas, a lack of training leading to knowledge deficit, poor cleaning techniques and staff incompetency. interventions included a checklist (audit tool) for environmental cleaning assessment of high-touch areas, a "train the trainer" programme for the iclns, an annual competency assessment and video tutorials on environmental hygiene to standardise practice. following the interventions, environmental cleaning assessment of high-touch areas showed an overall average of % compliance, with cb-dcs achieving ≥ % compliance in environmental cleaning of high-touch areas. this study illustrated that the intervention programme increased staff awareness, thereby improving compliance. besides promoting positive outcomes, it enhanced the internal monitoring system at nkf. antimicrobial resistance and infection control , (suppl ):e in march , a surge of mrsa acquisitions ( from screening and from urine cultures) was noted in rehabilitation ward (rehward) at tan tock seng hospital, singapore. our objective was to investigate if fomite transmission could be a cause of these acquisitions. we conducted one-day surveillance screening of the gym equipment and reh-ward's environment. samples were collected by rolling swabs moistened with sterile saline five times on the surfaces of gym equipment before and after use. in the ward, selected patients' beds and common items or equipment in shared area were also sampled. samples were cultured for mrsa using selective chromogenic media. in the gym, all samples collected from equipment pre-use were negative for mrsa. however, . % ( / ) of the samples collected after use were mrsa positive. in the wards, all swabs ( ) that were taken from the common shared area such as computers, case notes carts, were negative. two out of beds ( . %) occupied by mrsa carriers and out of beds ( . %) occupied by non-mrsa carriers were contaminated with mrsa (or . , %ci . - . , p = . ). overall rates of mrsa-positive swabs were comparable between the wards and gym ( . % vs. . %, p = . ). gym equipment was not more likely than the ward environment to contribute to mrsa acquisition. the importance of environmental cleaning in all areas including rehabilitation facilities cannot be overemphasised. with the widespread use of antibiotics in the treatment of human bacterial infections, the multidrug-resistant microorganisms also appear to threaten human health. environmental cleaning to avoid the spread of bacteria and healthcare-associated infection is an important part of healthcare infection control practice. through this program, our hospital aimed to improve the environmental cleaning, reduce the bacterial antibiotics resistance, and further reduce the use of antibiotics. we reformed program of environmental cleaning and measured incidence of multidrug-resistant bacteria and consumption of designated antibiotics. our results were shown below. the unqualified rate in environmental cleanliness of our cleaners was . % before this program was implemented and . % after this program that demonstrated . % reduction. the numbers of healthcare-associated infections with multidrugresistant bacteria was before this program, and after this program ( % reduction). the consumption of anti-methicillin-resistant staphylococcus aureus was . defined daily dose (ddd) / bed-days before this program, and . ddd / bed-days after this program ( . % reduction). the consumption of glycopeptides was . ddd / bed-days before this program, and . ddd / bed-days after this program ( . % reduction). the consumption of carbapenems was . ddd / bed-days before this program, and . ddd / bed-days after this program ( . % reduction). according to our results, environmental cleaning may effectively reduce the number of healthcare-associated infections with multidrugresistant bacteria and used of broad-spectrum antibiotics. if it can be promoted consistently, in accordance to the qualified data, the use of antibiotics could be reduced and the prevention of bacterial resistance occurred. antimicrobial resistance and infection control , (suppl ):e the invention related to an antibacterial treatment process of a curtain fabric material with functions of free washing and environmental protection. they were used for the antibacterial treatment of a nonwoven fabric made from polypropylene fibers and as the curtain fabric material with functions of free washing and environmental protection. before our hospital use this production, we aimed to investigate the expiry date of antimicrobial curtain to establish our protocol. we conducted this experiment from august to october . in this study, we used three companies' antimicrobial curtains. we used the c.difficile, mrsa and a. baumannii as study models. then we putted the antimicrobial curtain on the agar then see the bacteria growth or not. according to our study, c. difficile, mrsa and a. baumannii were not detected in the antimicrobial curtains. after weeks, there were no bacteria growths on the curtain. the curtain fabric material produced by the invention has the advantages of good air permeability, easy maintenance of dry curtain fabric surfaces, dirt resistance, free washing, no toxicity and irritative peculiar smell, easy recycling, environmental protection, good antibacterial property and low antibacterial treatment cost. antimicrobial resistance and infection control , (suppl ):e environmental contamination is the important source for bacterial spread causing hospital-acquired infections. environmental cleaning with an established standard operation procedure (sop) and cleaning staff's strict adherence to the sop are therefore extremely important. however, evaluation of the effects of environmental cleaning in general has not been fully reported in the literature. this study aim to elucidate the effects of environmental cleaning with the established sop and by the well trained cleaners in kaohsiung chang gung memorial hospital (kscgmh). environmental cleaning was based on the sop which followed the principle of cleaning from higher locations to lower ones, and from the contaminate areas to the comparatively cleaner ones in rooms where patients were staying. before and after daily environmental cleaning routine, environmental surfaces were swabbed for sampling specimens for bacterial culture and for bacterial count evaluation in case of culture positive. data showed that before and after cleaning environmental, bacterial burdens in environmental surfaces of the bedroom were significantly reduced (p < . ). indicating the environmental cleaning with the current cleaning sop and by these well trained cleaners has been effective. hospital-wide environmental cleaning monitoring program reduces healthcare-associated infections related to multidrugresistant organism hsuehlan chao, pinru changchien, weifang chen, chunghsu lai e-da hospital, kaohsiung, taiwan the evidence-based policies to clean hospital environment can reduce the colonization and infection of multidrug-resistant organisms (mdros). the purpose of this study is to measure the effectiveness of environmental cleaning policies. ( ) the effectiveness of policies was examined by adenosine triphosphate (atp) and microbial cultures (mcs) before and after the implementation of policies at both the general wards (gws) and the intensive care units(icus). ( )the study audited selected points related to mdros, including bed rails, bed button beds (the desktop corner), etc. ( )the standard values: atp less than rlu at icu and less than rlu at gw. bacterial colonies count < cfu. ( ) / atps ( %) had been detected over rul before policies, but / ( %) after policies without statistical difference (p = . ) in gws. however, in icus, before policies / atps ( %) over rlu and after policies / atps over rlu, no statistical difference (p = . ). ( ) % selected points were over rul both before and after policies at gws. only . % selected points were over rul in icus. ( ) mcs had statistically significant (p = . ) before and after policies at gws, including oxacillin-resistant staphylococcus aureus (orsa), enterococcus faecium (vre), acinetobacter baumannii, (xdr).but icus were not dirty over standard both before and after policies. this survey helps us understand how much dirty and contamination in the environment. especially, bed rails, button, isolation unit, car-related equipment, room telephone, curtains. establishing good environmental policies are very important to prevent healthcare-associated infections. icus environment is cleaner than gws in this study. antimicrobial resistance and infection control , (suppl ):h background contaminated hands are the foremost source of spreading infections in healthcare facilities. wellness and safety of patients and healthcare workers (hcws) can be achieved by promoting best practices in infection control through education and advocacy. we aimed to develop effective hand hygiene (hh) practices among hcws by improving their knowledge, attitude and practices through implementing standard hh guidelines. a year-long project was conducted at two hospitals of bangladesh. this included a baseline survey, intervention by implementing standard hh guidelines through classroom and hands-on training, and a post intervention survey. pretest-posttest was conducted with preformed questionnaire and observation checklist during pre and post intervention surveys. total of physicians and nurses were trained on standard hh practices. at the institute of child and mother health, rate of hh compliance before patient contact improved from % to . %(p < . ) among physicians and . % to % (p < . ) among nurses. after patient contact, it increased from . % to . % (p < . ) among physicians and . % to . % (p < . ) among nurses. at general hospital, sirajgonj, rate of compliance before patient contact increased from . % to . % (p < . ) among physicians and . % to . % (p < . ) among nurses. after patient contact, it increased from . % to . % (p < . ) among physicians and . % to . % (p < . ) among nurses. the project outcomes signify that implementing standard hh guidelines improves the knowledge, attitude and practices of the hcws. the results emphasize the necessity of continuous education and advocacy in improving hh compliance to promote excellence in infection prevention and control. antimicrobial resistance and infection control , (suppl ):h the world health organization multi-modal intervention to increase hand hygiene compliance was adopted in a rehabilitation hospital since . however, the effectiveness of the individual measure was unknown. this study was conducted for evaluating the effectiveness of hand hygiene promotion activities used in the hospital. a cross-sectional survey was applied in . a -item selfadministered questionnaire was adopted to collect the opinion on the effectiveness of measures previously used on hand hygiene promotion. nurses and healthcare assistants working in the inpatient settings of hospital were invited to participate in the survey. sevenpoint likert-type scale from "extremely ineffective" to "extremely effective" was used for ratingindividual items. rasch measurement was employed for data analysis by winsteps version . . . one hundred and seventy-nine questionnaires were returned contributing . % of the response rate. the categories in the rating scale were collapsed into -point scale before further analysis. thirtyone misfitting persons and three misfitting items were removed after examination in quantitative and qualitative manners. no differential item functioning was found between subgroups. the final scale was considered as unidimensional with reliabilities ranged at . - . and . - . for persons and items respectively. "placing the alcoholbased handrub" was identified as the most effective measure on hand hygiene promotion and "set up an annual target of hand hygiene compliance" was considered as the least effective. the survey identified and located the effective measures on hand hygiene promotion. for a more efficient approach, the hospital may prioritize the most effective items in hand hygiene promotion. antimicrobial resistance and infection control , (suppl ):h background adenosine-triphosphate (atp) bioluminescence assay has been popularly adopted in clinical and catering industry due to its ease of use and immediate results. atp bioluminescence assay picks up cellular discharged atp, which can also be found on cellular debris or organic components that are not microbial in nature.its measurement on animate objects can be misleading. this study was developed for the catering crew of a private hospital as a hand hygiene (hh) practice monitoring. microbial viable count was used as a validating reference for the atp relative light units (rlus) as a control measure of hh effectiveness. a set of basal microbial values was developed for each staff member. this provided a convenient but reliable protocol for atp luminometry users. swab sampling was collected from crews' hand for bacterial culture. selective media and serological tests were used for pathogen screenings, which included staphylococcus aureus, coliform and salmonella. standard curves to demonstrate the correlation between the viable microbial counts and the corresponding rlus of the atp measurements were developed. the study showed the actual viable microbial density of individuals after handwashing did not correlate positively with rlus. each individual had his/her own confidence regarding to the limitation of rlus. however, the hh compliance could be reflected by the viable microbial counts.individual skin condition played a role in this association. the measurement instilled a positive effect on the crew. hand hygiene compliance can be reflected with the bench marking standard technique. hand hygiene compliance was increased and microbial load was significantly reduced. hand hygiene is the single and most effective way to prevent the spread of microorganisms in hospital. when health care workers (hcws) have own sense and aware of the importance of hand hygiene, it yields twice the result with half the effort in infection control. the study aimed to promulgate hand hygiene is the responsibility of every hcw and maintain hand hygiene as the standard of care in the daily work of hcws materials and methods on the international hand hygiene day th may, a hand hygiene campaign was held in cmc. hcws were invited to take photo and pledged on hand hygiene compliance. hand hygiene technique was also taught personally by infection control nurses and return demonstration was needed during the activity. an instant photo was taken when they pledged. these photos were shown on a board during the hand hygiene promotion activity and returned to each colleague as a souvenir afterward. hospital managers, frontline doctors and nurses, allied health professionals and supporting staff, total over colleagues in cmc were pledged on hand hygiene on that day. this pledge motivated other colleagues to compile in hand hygiene. the hand hygiene compliance rate in cmc maintained over %. motivate health care workers to perform hand hygiene by a soft commitment is another way to promote and raise their awareness of hand hygiene. to introduce the "who hand hygiene save life campaign" and enhance the awareness of public and healthcare workers for the importance of hand hygiene. hong kong infection control nurse association (hkicna) has joined actively in the community events such as "world health day carnival" since . in these few years, there were over a thousand public to participate in hh and infection control related educational game booths and talks. further to extend the engagement of public and healthcare workers, a poster design competition for promotion of hand hygiene was organized in ; the winning poster was used as "talking wall" in community and healthcare settings and the background of hkicna's souvenirs. in , a creative reminder-hand-held electric fan with visual lit up "hand hygiene" was distributed. in addition, another innovative ideatwo hh dances were designed to continuously promote the hh; they stress that hh practice should start from children to adulthood, from healthcare worker to all in the community. both of them were used as a tool for promoting in hospitals and schools and assessable in youtube which was gained thousands of 'likes' conclusions hkicna is working hard on introducing hand hygiene concept for infections prevention and control in healthcare settings and community. the concept of "clean hand save lives" will continue be emphasized. antimicrobial resistance and infection control , (suppl ):h hand hygiene (hh) is recognized as the most effective measure to prevent the spread of micro-organisms through hand contact during patient care. besides yearly mandatory infection control training through on-line learning management system, matilda international hospital (mih) also provides relentless training to staff emphasizing the importance of proper hand hygiene and embracing who moments. in june , educational booth game -"unforgettable moments and steps" was held to assess knowledge and techniques of both clinical and non-clinical. this study aimed to evaluate the techniques of staff's hand hygiene practice, compliance to rubbing time and accuracy in reiterating the moments. observational methods were used to evaluate hh technique and rubbing time. staff were required to accurately call to remembrance the moments through direct questioning and demonstrate steps of hand hygiene technique with at least seconds of rubbing time. immediate feedbacks were supplemented. the overall accuracy of all assessed criteria's was . %. majority of hh steps achieved greater than % compliance rate except "between finger" and "back of finger". "before clean aseptic procedure" demonstrated to be the most difficult to recall. the audit allowed for gaps and in-depth understanding of staff hh practices to be more accurately identified with subsequent staff training strategies to be drawn and implemented. antimicrobial resistance and infection control , (suppl ):h background alcohol-based hand rubs (abhrs) are the preferred methods for performing routine hand hygiene (hh) in healthcare facilities. however, soap-and-water hand washing is still popular. this study measured the hh knowledge and self-reported practices of shiraz nemazee hospital nurses. this study employed two questionnaires. a six-question survey covered hh knowledge, ( possible points), while hh practices were monitored in a second survey containing four multi-part self-reporting inquiries ( possible points) in . surveys were voluntarily completed at work. responses were analyzed anonymously. results nurses completed the questionnaire. . %had formal hh training in the past year. . %reported using abhrs for more than a year. . % preferred traditional soap-and-water hand washing. eleven nurses never used abhrs. nursing experience varied - . % (>ten years), . % ( - years), . % ( - years) and . % (< years). knowledge scores ranged from - (high score was ). selfreported hh compliance scores ranged from - (high score of ). a positive, but weak correlation existed between knowledge and self-reported practice scores (r = . , p < . ). no correlation existed between years of experience and knowledge (p = . , r = . ) or self -reported practice scores (p = . , r = . ). also, no correlation was found between age and self-reported practices (p = . , r = - . ) and/or knowledge scores (p = . , r = - . ). antimicrobial resistance and infection control , (suppl ):h background simulation healthcare education is widely used in medical education and has great potential. however, scenario-based simulation healthcare education for preventing nosocomial infections has not been described. this study aimed to determine the effectiveness of scenario-based simulation education to improve hand hygiene. a single-centre, prospective, cohort study was conducted at tokyo medical university hospital ( beds), an acute-care teaching hospital, from january to december . each infection-control training course (ictc) was held every month and lasted hours. trainees put on and removed personal protective equipment under scenarios of standard precaution (two scenarios) and contact precaution with methicillin-resistant staphylococcus aureus (one scenario), while considering timing of hand hygiene. we determined the correlations between the participation rate in the simulation education and use of alcohol-based hand disinfection and reduction of catheter-related blood stream infection (crbsi). there were trainees. the total participation rate for hospital staff was % by the end of the study. the overall correlation between use of alcohol-based hand disinfection in the hospital and the course participation rate was significant (correlation coefficient, . ). an inverse correlation (− . ) was observed for the relation between the ictc participation rate and the incidence of crbsi. with participation in the ictc, crbsis due to staphylococcus spp. and enterobacteriaceae were significantly lower than those due to candida spp. our ictc had a positive effect on hand hygiene and reducing crbsi. this study is the first effective scenario-based simulation healthcare education to hand hygiene and control of nosocomial infection. antimicrobial resistance and infection control , (suppl ):h background the purpose of this study is to increase and sustain hand hygiene compliance through evidence-based approach and to relate compliance with the trend of healthcare associated infections in the hospital. the study was conducted over a year period from september to august . the methods used in monitoring hand hygiene compliance are "direct observation" which includes self-reporting and by the use of secret shoppers. another is "electronic monitoring" through the use of radio frequency identification device. surveillance of healthcare associated infection (hai) is conducted in the general nursing units, telemetry and intensive care units. interventions to increase hand hygiene compliance were implemented such as training of nurse linc representatives in monitoring hand hygiene compliance, use of social media (facebook) in promoting hand hygiene, recognition of individuals and department with high compliance. after the implementation of interventions, result showed an increase in the hand hygiene compliance from below %, during the start of the period monitored, to above % during the succeeding months. comparison of hand hygiene compliance versus healthcare associated infection rates was shown through a graph, this information was cascaded to the different departments during unit meetings. correlation showed a contrasting trend between hand hygiene compliance and healthcare associated infection rates. it was therefore concluded that an increase in the compliance to hand hygiene can decrease the healthcare associated infections among patients. in addition, feedback methods and other evidencedbased interventions can increase hand hygiene compliance. would also like to correlate the trend of hand hygiene rate with healthcare associated infections in the msicu. the data collected is generated by an automated system through the use of radio frequency badges worn by the healthcare worker and sensors attached to the hand rubs and soap dispensers. badges provide real time feedback by means of an alarm system. a quasi-experimental design was used to test the effectiveness of the interventions implemented. the utilization of visual boards in providing feedback, converting the door entrance into a giant hand hygiene poster, use of social media, and reward system were included in the interventions. total number of opportunities captured is , . post intervention data showed an increase in the compliance from below % to above %. it was therefore concluded that the multiple strategic approach, through the use of electronic monitoring, helped in increasing compliance of healthcare workers to hand hygiene. moreover, when hand hygiene trend was compared to the healthcare associated infection, a contrasting trend was evident. antimicrobial resistance and infection control , (suppl ):h hand hygiene (hh) is a known effective measure for the prevention of healthcare-associated infections and spread of antimicrobial resistant organisms. unfortunately a compliance rate with healthcare workers in emergency department is very low because of rushed working environment. the objective of this study focused on the interventions at the point of care (poc) to improve the compliance, or "system change" according to the who hand hygiene strategies, which key aspect was the provision of alcohol based hand rub on a new design bottle holder that optimized the acceptance and usage. the first phase was the baseline period set on months baseline (january to march ) observation of hh compliance and continue the second phase using the bottle holder (april to june ). an anonymous, self-administered questionnaire, had distributed to emergency healthcare workers (hcws) to assess their behaviors and attitudes toward hand hygiene compliance and their satisfaction. overall, . % ( of )of hcws ( nurses, physicians) satisfied with the device and . % believed they could improve hh compliance.overall compliance significantly increased from . % to % (p-valve = . ), to . % before patient contact, . % to . % after patient contact, . % to . % before clean/aseptic procedures, . % to . % after body fluid exposure/risk, and . % to . % after touching patient surroundings. the device successfully provide easy access to the bottle holder, which is critical for its success in improving hh compliance in one of the busiest area in the hospital. antimicrobial resistance and infection control , (suppl ):h hand hygiene compliance among hospitalized personnel is a one of safety indicator. in the past, our hospital had used the paper-based system for auditing hand hygiene compliance. the majority of the data entry, collection and analysis were performed manually which was complicated and time-consuming.we aimed to create an electronic hand hygiene auditing tool to measure hand hygiene compliance. the study was conducted in a tertiary-care hospital in thailand. a development process using five steps of the program development life cycle(pdlc). ) requirement gathering and analysis .)design on google docs access methods transition. .)program testing. .) implementation on mobile phones. .) maintain the program/system. all steps were conducted in a month (april ). the electronic hand hygiene auditing tool is the result of this study. both methods of data collection were compared. the results demonstrated that the duration of the processes decreased from months to month. questionnaires return rate increased from % to %.the assessors' satisfaction rate was %. antimicrobial resistance and infection control , (suppl ):h background good hand hygiene (hh) practices are a simple and cost-effective strategy to limit pathogen transmission between patients. this study explores the effect of a multimodal hand hygiene promotion program on hh compliance amongst healthcare workers. a prospective study was conducted at the pediatric intensive care unit (picu) and pediatric immunocompromised ward at king chulalongkorn memorial hospital, bangkok, thailand. interventions performed were: hh promotion videos sent to staff via mobile phone, hand hygiene signs at the bedside, distribution of portable alcohol gel bottles, and hh promotion culture led by senior staff members. all interventions were tailored according to pre-intervention opinion surveys with staff. hh compliance was assessed by direct observation using the who -moments for hand hygiene (who hh)before touching patients, before clean/aseptic procedures, after body fluid exposure risk, after touching patients, and after touching patient surroundings. opportunities in total were observed monthly. in december , pre-intervention, overall hh compliance rates were %. between january and june , post-intervention, overall hh compliance increased to %. when divided into the five moments for hand hygiene, hand washing prior to touching patients significantly improved following intervention from . % to . % (p < . ) on the immunocompromised ward and from . % to . % (p < . ) on the picu. hand hygiene after touching patient surroundings remained low. hh compliance was highest amongst nurses. a multimodal hh promotion campaign tailored towards the local population was effective in increasing hh compliance overall. however, hh after touching patient surroundings remained low post campaign. antimicrobial resistance and infection control , (suppl ):h the world health organization (who) has developed multimodal hand hygiene improvement strategies to support hospitals to improve hand hygiene and thus reduce hospital-acquired infection. the aim of the study was to study the implementation of these multimodal strategies, obstacles, and supports to promote hand hygiene in government hospitals including university hospitals, regional hospitals, and general hospitals. the samples were infection control nurses who implement multimodal hand hygiene improvement strategies. the data collection instrument was the hand hygiene self-assessment framework questionnaire developed by the who. the questionnaire also asked about obstacles and support. most of the samples ( . %) implemented multimodal hand hygiene improvement strategies at an intermediate level, followed by basic and advanced levels ( . % and . %, respectively). fifty percent of the university hospitals had an advanced level. most of the regional hospitals and the general hospitals ( . % and . % respectively) had an intermediate level. the highest score for implementation was the system change for hand hygiene (median score, . ), followed by reminders in the workplace, training and education, evaluation and feedback, and institutional safety climate for hand hygiene (median score, . , . , . and . , respectively). all samples encountered obstacles and needed support when implementing hand hygiene improvement programs. personnel were the first obstacle, followed by management, facilitative equipment, and budget, respectively. the greatest need was for environmental and equipment support, followed by support from staff and management support. the government hospitals should improve implementation of multimodal hand hygiene strategies by addressing obstacles and providing support in order to promote hand hygiene and reduce hospital-acquired infection. additionally, using e , the efficacy of these formulations was compared against seven commercially available abhrs and who recommended formulations containing alcohol from % to % at a more realistic -ml volume application. the novel abhr formulations met efficacy requirements for both hcphw and en when tested at volumes typically used in these methods. moreover, these formulations met hcphw requirements when tested at -ml. in contrast, the commercial abhrs and world health organization formulations failed to meet hcphw using a -ml application. importantly, product efficacy did not correlate with alcohol concentration and format. conclusions abhr are complex formulations, combining alcohol with various ingredients that can influence the overall antimicrobial efficacy. formulation as a whole and is more important than alcohol concentration alone for efficacy. product format has the same efficacy. when selecting abhr, ask for the in vivo efficacy results based on standard protocols and the volume of the product used to meet the criteria for hcphw test. antimicrobial resistance and infection control , (suppl ):he after the earthquake in kumamoto on april th, , disaster medical assistance team (dmat) and japan medical association team (jmat) immediately supported the shelters and medical facilities in the disaster area. however, the arrivals of dmat and jmat to nursing homes were delayed due to lack of information and no transportation by landslides. we started supporting nursing homes as jmat members days after the quake. this study aimed to report about our infection control intervention in the disaster area. we conducted a survey on the occurrences of infectious gastroenteritis and influenza at nursing homes, and interviewed the healthcare workers about problems on infection control. then, we offered guidance on infection control necessary to each facility. we visited eleven nursing homes between april th and may th, to find two residents with symptoms of influenza and one with symptoms of infectious gastroenteritis. the most frequent questions from the healthcare workers at nursing homes were how to clean and sterilize medical devices, and how to clean their hands appropriately. antimicrobial resistance and infection control , (suppl ):he as taiwan is situated in the high risk subtropical region, dengue fever has virtually become a seasonal infectious disease. this study aimed to determine risk factors and causes of death in dengue fever. analysis was conducted on the confirmed severe cases of dengue fever or dengue hemorrhagic fever reported to this hospital over the period between july and september , in terms of gender, age, history of chronic diseases, warning signs and diagnostic criteria for severe conditions. retrospective case study was also conducted to identify risk factors in dengue fever and dengue hemorrhagic fever as well as predictors of death among dengue fever cases for statistical analysis. antimicrobial resistance and infection control , (suppl ):he the importance of influenza is its rapid spreading of the epidemic in a wide range and serious complications, especially bacterial and viral pneumonia. taiwan medical and public health system experienced huge impacts of such epidemics. our study integrated epidemiological results to improve the effectiveness of influenza surveillance and assess the effectiveness of health policies for influenza prevention. we retrospectively reviewed recorded data of patients admitted in the intensive care unit who were diagnosed as severe influenza with respiratory failure during january , to december , . we also recorded their clinical outcomes and history of influenza vaccine administration. we analyzed their prognosis, predictors of death and their possible correlations. sensitivity analysis was performed to assess the severity of patients admitted in the icu. mechanical ventilation was estimated for epidemiological week of hospitalization. these patients had low proportion of treatment with antiviral medication in the first period of the study. influenza deaths were increased. it may contributed from aging of the population, underestimated the needs for better prevention modalities, including more effective vaccines and vaccination programs for elderly persons. ) . moreover, the length of stay in the hospital of the study group was significantly higher than those in the control group (p < . ). controlling and preventing cre infection in medical ward and surgical ward were important and necessary. especially among patients who were admitted in the hospital for longer than weeks. antimicrobial resistance and infection control , (suppl ):he background newborn could be seen as patients who are potentially exposed to ventilator associated pneumonia (vap) at high risk. identify the incidence as well as risk factors for vap in newborn would be much necessary for prevention application. this study aimed to identify incidence and risk factors of vap in nicu at the national children's hospital (nch) in vietnam. this cohort prospective study was conducted between january and december in . after univariate analysis, multivariate regression analysis was used to handle all statistically significant risk factors. and bactec fx were used to identify the total blood volume that was obtained. an average blood volume was . ml. and the positive rate of blood culture was . %. the emergency room had a lowest mean blood volume ( . ml) with a positive blood culture rate of . %. the surgical intensive care unit had a highest mean blood volume ( . ml) with a positive blood culture rate . %. in addition to a number of blood culture set, amount of blood taken for blood culture plays an important role for the positive rate. according to the clsi m blood culture guidelines, the recommended blood volume taken from adult is at least ml per bottle. the recommended blood volume from the blood bottle company is - ml. adherence to the clsi recommendation could improve the positive rate, which would result in reduction of antibiotic overuse, reduction of hospital expenditure as well as improvement of quality of care. this study was conducted between march and october . a total of stool samples were transferred using % etoh as a transport buffer and were processed by using the chromagar as a selective media. toxin screening was performed by using the multiple step eia kit as. finally, we used the pcr methods to validate the final result. before this process, the culture positive ratio was very low ( %) in our hospital. the cdi ratio is only / , patients while the cdi ratio of other medical in taiwan is more than / , patients. after implementation the new diagnostic scheme, the culture positive ratio increased to % and the cdi ratio was higher than before. we also performed the gdh eia test which revealed the similar results to the culture methods. our study confirmed that the gdh eia may be an alternative option for diagnosis of cdi. furthermore, use of % etoh as a transport media would improve the cdi ratio. antimicrobial resistance and infection control , (suppl ):m background chlorhexidine gluconate (chg) is a cationic bis-biquanide biocide with low mammalian toxicity and broad-spectrum antibacterial activity. positively charge of chg molecules bind to negatively charge of phospholipid in bacterial cell wall coursing membrane disruption. incompatible of anionic thickener in lubricating gel formulation can negate the efficacy benefits of chg. this study was carried out to investigate the in vitro chemical and biological incompatibilities of marketed lubricating gels with chg solution. five marketed lubricating gels containing anionic thickener (carbomer) and two marketed lubricating gels containing nonionic thickener were collected to investigate for in vitro kinetic chemical and biological incompatibilities with %w/v chg in water. the contact time was , , , , , and seconds. the results demonstrated that, five marketed lubricating gels containing carbomer reduced the amount of chg ( . - . %), which was measured by reversed-phase hplc technique and other two marketed lubricating gels containing nonionic thickener maintained the amount of chg. the antimicrobial activity revealed that %w/v chg solution achieved the . and . log reduction for staphylococcus aureus atcc and escherichia coli atcc , respectively. the antimicrobial activity was dramatically reduced to the . - . log reduction for s. aureus atcc and . - . log reduction for e. coli atcc after seconds mixed with five marketed lubricating gels containing carbomer. two marketed containing nonionic thickener maintained the antimicrobial activity of %w/v chg solution. it can conclude here that, anionic thickeners in lubricating gel formulation may neutralize positively charge and reduce the antibacterial activity of chg. mobile phone serves as a major reservoir of pathogenic microbes and may act as major carriers for transmission of pathogens from laboratories. the aim of the study is to determine pathogenic bacterial contamination on mobile phones using by medical technology students in clinical microbiology laboratory practices. bacterial contamination were isolated and identified from overall surface of mobile phones after touching by medical technology students before and during clinical laboratory microbiology practice. species of pathogen contaminated on mobile phones used by % ( from ) of students were identical with the pathogenic organisms that were given for practice in each student. a total of bacterial isolates were found on student's mobile phones collected before practice. these were s. epidermidis %, bacillus spp. %, mssa %, gnf %, s. saprophyticus %, micrococus spp. %, enterococcusspp. %. sample collected during clinical microbiology practice found bacterial isolates these isolates were identified as bacillus spp. %, s. epidermidis %, mssa %, gnf %, p. vulgaris %, k. pneumoniae %, e. cloacea %, e. aerogenes %, s. rubidaeae %, s. saprophyticus %, s. marcescens %, p. agglomerans %, c. freundii %, s. paratyphi a %. significant higher numbers of bacterial isolates (p < . ) obtained from student's mobile phones during clinical microbiology laboratory practice were observed in comparison with those obtained before practice. these results indicated that mobile phone can act as a mobile reservoir for laboratory and hospital infection. therefore, mobile phone using should be prohibited during clinical laboratory practice. in the additional; mobile phone decontamination and hand washing; should be strictly included as essential tools for laboratory safety and infection control. antimicrobial resistance and infection control , (suppl ):nt background universal prophylaxis and preemptive therapy are used to prevent cytomegalovirus (cmv) disease after transplantation. however, the optimal strategy has not been identified. this study aimed to evaluate optimal prevention strategies, particularly in high-risk recipients (donor-positive/recipient-negative [d+/r-] cmv serostatus pairs). we studied recipients ( high-risk, intermediate-risk; median follow-up > , days). asymptomatic cmv infection and cmv disease developed significantly more frequently in high-risk than in intermediate-risk patients ( . % vs. . %, p = . ; . % vs. . %, p < . , respectively). a higher peak cmv antigenemia titer ( vs. cells, p < . ) and a longer duration of antigenemia ( vs. days, p = . ) were observed among high-risk patients. among high-risk patients, all cmv infection and cmv disease cases developed within the first months and months, respectively, after transplantation. survival analysis showed no significant difference in mortality rates (log-rank p = . ) and graft loss rates (log-rank p = . ) between the groups. graft rejection occurred more frequently among high-risk patients, but the difference was not significant (log-rank p = . ). graft rejection was significantly more common in patients who developed cmv infection/disease than in those who did not (logrank p < . ), regardless of cmv serostatus. further studies comparing universal prophylaxis and preemptive therapy, particularly in high-risk patients, are required to identify the optimal strategy to reduce graft rejection rates. background japan is a natural disaster-prone archipelago. however few guidelines for public health activities have focused on infectious disease outbreaks during disasters. we have created the risk management guideline for infectious diseases during disasters to develop a comprehensive support system for prevention of infectious disease outbreaks in disasters. interviews were conducted with fifteen nurses who worked in any disasters occurred since the great hanshin-awaji earthquake in . the interview data as well as published documents were analyzed. factors influencing infectious disease outbreaks were extracted as categories by six researchers. furthermore, measures were extracted while universalizing these measures. following categories were extracted: "restroom", "water management", "hygiene products", "hygiene behavior to be clean" "environment", "food hygiene", and "situations of infectious diseases". there were measures in "restroom", measures in "water management", measures in "hygiene products", measures in "hygiene behavior to be clean", measures in "environment", measures in "food hygiene", measures in "situations of infectious diseases". measures could be visualized as a risk management guideline for infectious diseases outbreaks. this guideline is regarded as useful for preparing future disasters. however, it is necessary to consider that measures vary according to what kind of, where, and when disasters occur as the guideline was extracted from disaster experiences in japan. the rapid emergence of resistant bacteria is occurring worldwide. the prevalence of antibiotic resistance among the aforementioned bacteria has been increasing. this study was carried out to develop novel formulation against antibiotic-resistance microbes using for cross-transmission and cross-contamination control. the novel formulation was developed using quaternary ammonium compounds as major active ingredient and synergistic with three minor active ingredients. the antimicrobial activity against antibioticresistance microbes, methicillin-resistant s. aureus it can conclude here that, the novel formulation exhibited the potent antimicrobial activity against antibiotic-resistance microbes and also showed the safety in animal study. microbiology laboratory isolated ralstonia picketti in blood cultures from four patients who underwent hemodialysis on the same day at a same center, similar antibiogram of all these isolates lead to a suspicion of an outbreak. repeated blood cultures and the jugular venous catheter tip from one of the patient grew the same pathogen leading to the suspicion of this patient as the index case of the outbreak. routine cleaning, disinfection and sterilization of the water treatment system did not stop further patients from experiencing rigors and infection with the same pathogen. no new patient was identified after the cessation of dialysis reprocessing in the center. this confirms ralstonia pickettii has infected the dialysis reprocessing system from the index case that was dialyzing via a jugular catheter, and subsequently, infecting other patients in the center. ralstonia pickettii can be encountered in dialysis facility through contamination of water treatment and reprocessing system, resulting in outbreaks. early identification of pathogen is essential to prevent escalation of outbreaks in dialysis centers. antimicrobial resistance and infection control , (suppl ):oi klebsiella pneumoniae are important pathogen in healthcareassociated infections. therefore, carbapenem-resistant klebsiella pneumonia (crkp) causes infections with high mortality rates, which the ratio of increases in the recent years. this study aimed to investigate the epidemiological characteristics of crkp in a tertiary hospital in beijing and seek for control strategy. retrospective survey and temporal-spatial distribution analysis were used to crkp cases newly diagnosed from jan to april, and pulsed-field gel electrophoresis (pfge) was used to analyze the homology of crkp isolates. in the crkp cases, the median duration of hospitalization within days before detected was days ( - days), the duration of using carbapenems was a median of days ( - days), cases had history inpatient of other hospitals before admission. the major sources of specimens with crkp positive were blood, respiratory tract and urinary tract, with a positive rate of %, % and %, respectively. homology test was performed in crkp cases from wards. mlst analysis showed that cases were all st subtype producing kpc- enzyme; pfge results showed that cases were type , cases were type , with unclassified. temporal-spatial distribution of type and type cases suggested that there might be contact transmission between cases. it is necessary to establish regional multidrug-resistant organisms monitoring and tracking network and information should be shared between departments and hospitals during patient referral, which was the key strategy for crkp prevention and control. cgmlst, wgmlst and pangenome typing showed ultimate discriminatory power. concordance between different typing methods were poor at type level, excluding wgmlst-cgmlst. on cluster level, the agreement was better achieving % and more. antimicrobial resistance and infection control , (suppl ):oi background multilocus sequence typing (mlst) as a genotyping methods has been extensively used for study of evolution and population dynamics of s.aureus. the knowledge about genetic distances of genomes within and between mlst types estimated on wgmlst can be useful for understanding the population structure.we explored the variation within and between mlst types of global collection of s.aureus using wgmlst typing. materials and methods s.aureus genomes from genbank were analyzed with seqsphere + to determine mlst and wgmlst types. data with more than % missing values were excluded from analysis. genetic distance was calculated with nei formula (neim., ) using wgmlst data with loci. eight mlst types including s.aureus were determined: st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), st (n = ) and st (n = ). the mean of variation within mlst type was . % ( %ci; . - . ). in some sts, such as st , st , st , the genetic distance reaches . %, . % and %, respectively. in general, the difference between isolates from different sts ranged - %. however, in some cases, the difference between s. aureus from different mlst types were much lower - % (st -st ), % (st -st ), % (st -st ) and % (st -st ). analysis of global collection s.aureus isolated from different sources and places demonstrated high genome difference between mlst types differing by alleles. while genetic distances within and between closest sts might be overlapped what makes impossible to find appropriate cut-off for s.aureus clustering using wgmlst data to differentiate mlst with slv adequately, at least in some cases. antimicrobial resistance and infection control , (suppl ):oi in june , three nurses had flu-like symptoms in our medical, surgical and neurological wards within couple days. we started outbreak investigation in medical, surgical and neurological wards. we identified nurses with influenza a infections. after investigation, we found that compliance of wearing surgical masks while caring patients among healthcare providers is adequate but the compliance of hand hygiene is inadequate. furthermore, they neglected their flulike symptoms and did to practice good respiratory hygiene or cough etiquette. although these healthcare providers are able to follow the infection control standards while taking care a patient, they fail to follow the rule while contacting with staff members. these resulted in outbreak among healthcare providers. the outbreak was successfully controlled by introducing fever surveillance, advocating respiratory hygiene and cough etiquette and reinforcing environmental clearness. of these findings, enhanced hand hygiene and associated infection control strategies should be implemented to effectively reduce the transmission of type a influenza. background this research and development was a cohort study from to . firstly, varicella disease had impacts on hospital administration: ) unable the ward having patient(s) who had varicella infections to admit new patients, ) infected non-immunized hospital staff. secondly, the disease interfered normal hospital services: ) the affected ward was unable to admit new patients for months because it contained the infected patients ) an affected ward increased amount of new patients in others under limited resources of staff and facilities. in consequences, it increased patients' complains on hospital services. this study aimed to develop a guideline for controlling an outbreak of chickenpox in in-patient department and to evaluate the use of this guideline. this study was separated into stages: ) isolating patients having varicella infections, prohibiting admission of new patients in the units and evaluated contact persons by testing for varicella immunoglobulin g (igg), ) isolating patients having varicella infections but allowed admission of new patients into the units, evaluated contact persons by testing for varicella igg and used protective isolation room for patients who were at risk for acquiring the disease and )isolating patients having varicella infections, allowed admission of new patients, evaluated contact persons by testing for varicella igg, used protective isolation room for high-risk patients and vaccinated individuals who had negative results for varicella igg. no additional patients having varicella infection were found even though the hospital did not stop accepting new patients. the study is in progress to create screening criteria to identify patients having varicella infection before hospitalization. infection control personnel had identified this event through routine surveillance, and implemented some infection control measures immediately. the interventions included the following points: ( ) cohort care for the infected patients was introduced; ( ) daily environmental cleaning with ppm bleach was implemented; ( ) the other patients were followed with active screening for crkp colonization; ( ) environment and healthcare workers were screened for crkp colonization; ( ) healthcare professionals were educated for contact precautions and hand hygiene. among swab cultures from rcc environment, crkp were isolated from a bedside table and a physiological monitor of rcc and an infusion pump of rcc . we collected samples of hcws' hands and found crkp from one nurse, who was a key nursing staff of rcc patient. anal swab screening in contact patients showed two of crkp colonization that contacted withrcc and rcc patients. the study showed that poor compliance of hand hygiene among staff and wards' environment contamination was the reasons for this outbreak. the implementation of hand hygiene and ward environment cleaning and disinfection along with other infection control interventions were very important. however, this incident has not yet been ceased. ongoing monitoring and enforcement of infection control measures are still required. background incidence of clostridium difficile infection (cdi) in taiwan has not been widely studied, because the yield of culture based diagnostic test was low and the clinical characteristics of cdi had limited studies in the past. however many studies found the incidence of cdi increased in recent years. this study aimed to analysis on patients clostridium difficile infection to prevent cross infection. the following data was retrospectively reviewed; clinical evaluation, operational definition/diagnosis, diagnostic test using rapid molecular detection of toxigenic c. difficile. the medical records was reviewed and collected in case assessment form, which included underlying chronic diseases, past medical history, history of hospitalization, recent operation, past and current medication, particularly antibiotics use, and nutritional status. one-year incidence in our hospital was . %. of the patients with cdi, patients underwent chemotherapy, patient had intestinal surgery, patients were on hemodialysis, and patients had diabetes mellitus and long-term use of steroids. all patients were hospitalized within days, and received antibiotics. the medication duration was - weeks. the hospitalization time was - days. the mean age of patients was years old. the development of rapid and reliable diagnostic tools is important to identify cdi. effective treatment, prevention strategies are needed to control this rising infection. antimicrobial resistance and infection control , (suppl ):oi in september , the infection control nurse at a tertiary care hospital, northern thailand was notified of an influenza-like illness (ili) outbreak among the health care workers (hcws) in a medical record department. we conducted an outbreak investigation to determine the etiologic agent, identify additional cases, and implement control measures. an outbreak investigation of respiratory infections among hcws in a medical record department was promptly initiated. hcws with influenza ili were enrolled during august -september . respiratory swabs were collected fromthe hcws with ili andwere tested by realtime reverse transcriptase polymerase chain reaction (rt-pcr) for respiratory pathogens. mycoplasma pneumoniae is a significant cause of respiratory disease in this outbreak. the infection control team alerted hcws to the outbreak and recommended prevention measures. mycoplasma pneumoniae should be considered as a possible cause of respiratory disease in hcws in outbreak setting. morbidity from outbreaks in hospital might be minimized if facilities closely monitor for respiratory disease clusters, promptly report outbreaks to infection control team, prioritize diagnostic testing in outbreak situations, and timely implement strict infection control measures to halt transmission. background surgical site infections (ssi) following coronary artery bypass graft (cabg) procedures is a devastating complication with significant morbidity, mortality, and extra-cost. this study aims to describe the incidence of ssi and causative pathogens following cabg surgery in a university hospital during - . hospital records and post-discharge surveillance data between and were reviewed. ssi was diagnosed according to the us-cdc's surveillance definitions. there were patients undergone cabg surgery. of these, ( . %) had ssi; at sternal sites and at leg harvest sites; classified as superficial ssi and as deep ssi. this prevalence is higher than what have been reported. in this study, proportion of grampositive and gram-negative bacteria was % each. of cases with ssi, cases had polymicrobial infection. gram-positive bacteria included coagulase negative staphylococci . %, enterococcus spp. . %, and staphylococcus aureus . %. gram-negative bacteria were escherichia coli . %, klebsiella pneumoniae . %, enterobacter cloacae . % and pseudomonas aeruginosa . %. there was no difference in the prevalence of infection among surgeons. one-third of patients received prophylactic antibiotic very closely to operation, (≤ minutes) and it was discontinued mostly within hours after surgery, while . % of all patients received antibiotic for longer than hours. post cabg ssi was high in this study, probably related to inappropriate administration of prophylactic antibiotic. in-depth study is needed to delineate other risk factors which could affect the implementation of preventive strategies. the -dose vial (with preservative) of pneumococcal non-typeable haemophilus influenzae protein d-conjugate vaccine (phid-cv, gsk vaccines) was developed to improve logistics of and adherence to immunization programmes. the aim of this study was to assess reactogenicity and safety of the investigational phid-cv -dose vial presentation in infants. in this phase iii, mono-centre, observer-blind study (nct ) conducted in bangladesh, - -week-old infants, randomized : , received primary vaccination with either phid-cv -dose ( -dose group) or phid-cv -dose vial (preservative-free, -dose group) at / / weeks of age. dtpw-hbv/hib and polio vaccines were (co)-administered at / weeks of age. solicited and unsolicited adverse events (aes) within and days post-vaccination, respectively, and serious aes (saes) throughout the study were assessed. the total vaccinated cohort comprised infants in each group. the most commonly reported phid-cv injection site and general solicited aes were pain (after . % and . % of doses in -dose and -dose groups, respectively) and irritability/fussiness (after . % and . % of doses, respectively). the overall/dose incidences of other injection site (redness and swelling; . %- . %) and general aes (drowsiness, loss of appetite and fever [axillary temperature ≥ . °c]; . %- . %) were within similar ranges between groups. unsolicited aes were reported after . % and . % of doses, and saes were reported for and infants in -dose and -dose groups, respectively, none was considered as vaccination-related. two infants died (sepsis, -dose; septic shock, -dose). phid-cv -dosevial presentation (with preservative) had an acceptable reactogenicity and safety profile, similar to phid-cv -dose vial (preservative-free) in infants. immunogenicity of -dose vial presentation of pneumococcal non- antimicrobial resistance and infection control , (suppl ):o to facilitate multi-dose use, gsk vaccines developed the pneumococcal non-typeable haemophilus influenzae protein d-conjugate vaccine (phid-cv) -dose vial, which contains preservative. the aim of this study was to demonstrate non-inferiority of the immunogenicity of investigational phid-cv -dose versus licensed -dose vial presentation in infants. in this phase iii, observer-blind study (nct ) conducted in bangladesh, - -week-old infants, randomized : , received phid-cv primary vaccination at ages / / weeks with either -dose ( -dose group) or -dosevial (preservative-free, -dose group). dtpw-hbv/hib and polio vaccines were (co)-administered at ages / weeks. immune responses (antibodies against pneumococcal serotypes [ f-elisa] and opsonophagocytic activity [opa]; anti-protein d antibodies [elisa]) were measured. non-inferiority of phid-cv -dose versus -dose for each vaccine pneumococcal serotype (vt) and vaccine-related serotype a (confirmatory objectives) in terms of antibody geometric mean concentration (gmc) ratios was assessed. of vaccinees, ( -dose) and ( -dose) were included in the according-to-protocol cohort for immunogenicity. non-inferiority criterion (upper limit of -sided % confidence interval of the antibody gmc ratios [ -dose/ -dose] < -fold) was met for each vt and a. for each vt, ≥ . % of infants in each group had antibody concentrations ≥ . μg/ml, except for b ( . %, -dose; . %, -dose) and f ( . %, -dose; . %, -dose); for a, ≥ . % of infants in both groups. opa for each vt and a, and anti-protein d responses were within similar ranges between groups. immunogenicity of phid-cv -dose vial (with preservative) was noninferior to -dose vial (preservative-free) in terms of antibody gmc ratios for each vt and a post-primary vaccination in infants. percutaneous injuries resulting from contaminated sharp devices among healthcare workers (hcws) is a major concern of staff safety in hospitals, with increased risk hiv, hepatitis b and c infections. percutaneous injuries were at top five risks in our hospital in - , with > % of injuries related to operating theatre (ot). we aimed to study the impact of a multi-interventional strategy on incidence of percutaneous injuries at a non-profit private care hospital in hong kong. canossa hospital (caritas) is a -bed non-profit private care hospital treating approximately , patients yearly with hcws. between - incidents of percutaneous injury occurred annually in - . from , a multi-interventional strategy was introduced to reduce incidence of percutaneous injuries, including: identifying risks of bloodborne infections exposure, formulation of ward policy guidelines, staff education and training to create safe workplace environment, enhancement of reporting system, individual counseling by infection control nurse, provision of safety-engineered sharps devices (e.g. needle counter in ots), verbal-visual reminder (meetings, posters). compliance audits were performed in ots after implementation. number of percutaneous injuries declined from between - incidents in - to and incidents in and respectively. the proportion of injured staff showed an initial decrease of % (p = . , chi-square test) in and then significant reduction of % (p < . ) in . we achieved reduction in percutaneous injuries using multiinterventional strategy. implementation of guideline formulation, education, individual counselling after incident, verbal-visual reminders, safety devices and performance audits led to an improvement in hospital staff safety. surgical ward ( cases). during this study period, . % cases occurred sharp injury in relation to blood taking procedure (n = ). waste collection is the second commonest situation for sharp injury at . % of reports (n = ) and cases reported during disposing sharps into sharp bins ( . %). doctors reported the highest incident with cases ( . %) while nurses reported cases ( . %) and various groups of hcws constitutes the remaining . %. all hcws are at risk of sharp injury however certain groups are at higher risk. the rate of sharp injury is persistently high despite the ongoing education program for hcws in our institution. background influenza can be a serious disease, since health care workers may care for or live with people at high risk for influenza-related complications. this study aimed to increase staff vaccination rates in and to know whether the additional cost is serving its worth, which shows the incremental cost -benefit ratio of increasing vaccine coverage. in , the team conducted unit by unit information campaigns through floor to floor dissemination of information about free vaccination in the employee health clinic, and lastly "i am asian" jackets were provided to staff who underwent the vaccination. in from to , the infection prevention and control office conducted an outbreak investigation on the cases of vzv among healthcare workers. this involved contact tracing and review of medical history of the healthcare workers. a survey on the immunization history of all healthcare workers (hcw) was done. outbreak control was also recommended through vaccination of all exposed that have no active immunity on the virus. the calculated cost of the treatment for hcws was done. result of the investigation revealed that % of the cases are cross transmission from one healthcare worker to his other co-workers. most of the healthcare workers who are on the prodromal stage of the virus still reported to work. the cost of the treatment per personnel who have vzv was calculated at , php ( usd) while the cost of the vzv is at , php ( usd). among all hcws % have no active immunity to vzv while % have no vaccination to vzv. there is a need to strengthen vzv information campaign among hcw. mandatory immunization of all susceptible staff to vzv must be recommended and barriers to vaccination uptake among hcws must be studied. reiteration of the sick leave policy must be emphasized. o nursing experience with one postoperative oral cancer patient using transdisciplinary care model ching-i ting (alice @yahoo.com.tw) chi-mei medical center intensive care unit, tainan, taiwan antimicrobial resistance and infection control , (suppl ):o oral cancer patients are susceptible to postoperative structural defects of tissues such as in the orofacial region and jaw bones, which cause changes in the facial appearance, defects of physical functions such as dysphagia, slurred speech and eating difficulties and consequent negative impacts on quality of life. each of these patients, therefore, represents a highly complicated case with multiple problems and requires collaboration between a wider range of specialty departments on case discussion and provision of care to reduce their postoperative complications and improve their quality of life. the healthcare provided to the patient and the family through the transdisciplinary care model included: ( ) alleviation of wound pain for the patient; ( ) planning of training sessions for rehab from dysphagia; and ( ) continuing coaching for the patient's mother on caregiving for the patients at home. this paper, based on the reason for and the definition of transdisciplinary care, presents for the reference of clinical practitioners leaders of medical institutions a specifically addressed strategy of evidence-based transdisciplinary practice, integrating the care plans suggested by all the teams and providing healthcare and rehab plan that reduced and improved the patient's wound pain and dysphasia. the evidence-based clinical practice of transdisciplinary care is a key trend of care in medical environments. using transdisciplinary evidence-based care as the foundation helps healthcare professionals across disciplines to understand and respect the spirit of collaborative care and obtain best treatment strategies through communications, thereby providing good quality of care. the introduction of asean economic community (aec) and thai government's policy in creating an international center for medical treatment, there has been a significant increase in number of patients travelling into thailand for treatments. patients are travelling on commercial aircrafts as well as air ambulance when their conditions are urgent or critical. air ambulance is a well-recognized method of transportation by the insurance company which medical wings has been serving patient from around the world since . at medical wings, we focus on safety, quality care and infection control. on occasion, we receive patient from facility which is not able to adequately assess infectious disease so our medical team are the first group to meet patient and preparation is the utmost important in a successful mission. we aimed to prevent the exposure of patients, visitors and healthcare workers to communicable or infectious diseases by stressing maintenance of sound habits in personal hygiene and individual responsibility in infection control, monitoring and investigating potentially harmful infectious exposures, providing care to personnel for work-related illnesses, identifying infection risks related to employment and instituting appropriate preventive measures. this study was performed between october st, and march st, . the imu had beds, were single-bed isolation room but the remaining was in a common hallway divided into three -bed sections. space between each bed was only cm. a total of patients were observed. after the first three months to determine the baseline prevalence, we implemented the control measures including cohorting patients with xdr-ab in the same area, encouraging hand hygiene by providing alcohol hand rub solution at each bed and common areas in the ward, mandatory isolation gown and gloves for caring of patient with xdr-ab, improving environmental cleaning technique and frequency. the prevalence of xdr-ab was gradually decreased, from . episodes / , patient-days in the first phase to . episodes / , patient-days in the second phase. this study suggests that cohorting along with stringent infection prevention technique was effective in controlling spread of drugresistant organisms and it might be an option for the crowded patient care areas. descriptive statistics were used to analyze the data. the adherence rate to gloving was highest, followed by gowning and hand hygiene ( %, % and % respectively). hand hygiene was performed more after than before giving care to patients. although they wore gowns and gloves before patient care, hcws immediately removed gown and gloves infrequently afterward. forty percent of the hcws adhered to the guidelines in every step, whereas % did not adhere at all. nurses adhered most to the guidelines ( %). hcws agreed with the guidelines but were unable to adhere to the guidelines at every step because of the crowded environment and perceived work overload. results of this study suggested that adherence to the guidelines for contact precautions needs more cooperation among hcws and barriers to the adherence should be minimized to prevent widespread multi-drug resistant organisms, and thereby improving the quality of care. the gloves compliance rate was highest than gown and hand hygiene. overall compliance rate %. the study finding needs to be concerned by healthcare workers for improving the quality of care. the methods of study were used development and empowerment program (dep), which followed role and competency of advanced practice nurse (apn). data were gathered from medical and surgical departments from january to september . there were purposive selected groups: ) case manager team, ) medical and surgical nursing teams and ) xdr infected cases. the research tools were: ) dep, ) data questionnaires, ) the xdr ic practice observational forms and ) the evaluation quality of xdr cases forms. data were analyzed using descriptive statistics and t-test. the results of study suggested that a model for nursing care should consisted of components: ) providing xdr infection control activities in process of care, ) developed and empowered case managers in role and competency of apn and ) feedback outcomes of caring and supporting system. after using dep, the xdr infection rate significantly decreased from . to . per patient-days (p < . ). this study showed that using dep in case managers and nurses promoted participants learning in teams. the dep also aligned xdr caring processes with ic standard practices that resulted in decreased xdr infection rate in hospital efficiency. background transmission of multidrug-resistant organisms in hospitals has a direct impact on patients, health care personnel, the hospital, community, and the nation. this developmental research aimed to develop a clinical pathway for prevention and control of multidrug-resistant organism transmission in the medical department at a regional hospital. study samples included health care personnel who worked in the medical department and related including: hemodialysis, echocardiogram, radiology and ultrasound, ct scan, mri, and stretcher. ten patients infected or suspected of multidrug-resistant organism infection at the medical department were included in the test. the process for developing was based on cheah's ( ) framework. three development steps were thus included: assessment and situation analysis, designing, and testing the implementation. the data collection instruments consisted of a demographic data record form and questionnaire assessing the opinions of health care personnel towards. the content validity of the questionnaire was examined by experts and the content validity index was . . data were analyzed using descriptive statistics and data categorization. the we demonstrated that our education-based intervention program was effective to reduce the rate of lcbi among clbsi. this is primarily due to the lowering the risk of contamination of common commensal in the blood specimen. improved cauti data collection methods idc reminder system nurse-led protocol to empower nurses to remove catheters in simple cases using s hook to keep urine bag below bladder level during ambulation daily maintenance audit of idc care frontline staffs were regularly engaged and feedback gathered. the changes were modified, tested and improved after (plan-do-study-act) pdsa cycles. preliminary results in the pilot wing showed a reduction in cauti rate although it is still early to tell if results are sustainable. results on compliance to idc care have also been encouraging. we plan to test and spread the changes to other wards to reduce cauti rate in the hospital. getting feedback and buy-in from the ground helps in designing sustainable changes. antimicrobial resistance and infection control , (suppl ):p background ventilator-associated pneumonia (vap) with vap bundles, should result in dramatic reductions in the incidence of vap. compliance with the vap bundle has been most successful when all elements are executed together as a \"all or none\" strategy. vap increases patient mortality, hospital stay and medical expenses. according to the latest vap criteria of the american disease control agency, there are many difficulties and blind spots in the diagnosis of vap, and the low rate of vap in this hospital is expected to be improved by the study of vap low reporting rate. the study of the hospital vap low reporting rate of the underlying causes: first, the clinical symptoms and chest x-ray interpretation of the lack of uniform standards, lack of education and training and advocacy. second, the existing receiving process can not immediately grasp the patient condition. third, to strengthen the quality of x-ray film and rules to track. the team developed the criteria and reporting process for the diagnosis of high-risk vap patients by the centers for disease control, in order to facilitate vap bundle in the care of patients with high-risk vap infection. the rate of vap in our hospital increased from ‰ to ‰, but it was significantly lower than that of foreign vap. difficulty in diagnosing patients and blind spots in vap are also a major limitation for clinicians. of vaps have a low reporting rate. therefore, the proposed medical institutions to standardize the hospital medical behavior and infection control measures to cohesion team consensus. the cvc bundle was implemented on all patients admitted to the icu starting april . data of crbsi rates from and were pooled to compare the differences between observed values and predicted values before and after the intervention. cvc bundle care was implemented since april . our study showed that the central catheter bloodstream infection rate was . ‰ (p = . ), the ventilator-associated pneumonia (vap) infection rate was . ‰ (p = . ), the catheter associated urinary tract (ca-uti) infection rate was . ‰ (p = . ), and the icu infection rate was . ‰ (p = . ). the overall central catheter utilization rate was . % (p = . ); the mean length of icu (intensive care unit) stay was . days, and the average length of a hospital stay was . days. the time sequence analysis model was adopted to analyze the catheter-related bloodstream infection rate in . although it did not decrease, but only slowed down the trend of increase the crbsi incidence. to effectively reduce the catheter-related bloodstream infection rate is to persistently support the combination of cvc bundle care and to enhance the compliance of the policy. the clinical benefit of chlorhexidine gel in severe units was the use of chlorexidine gel containing chlorhexidine mouthwash compared with the use of oral disinfection can be reduced by about , yuan. nursing care hours, about . hours per month can be reduced. the density of vap was maintained at . ‰. the use of chlorhexidine gel for mouth disinfection % less than the cost of mouthwash and save % of the time, not only reduce the cost of medical expenses also save the number of clinical nursing work, effectively improve the medical quality. to investigate the feasibility and compliance of the male catheterization in clinical practice yan-ru chen, yu-hsiu lin chi mei medical center,liouying, tainan, taiwan correspondence: yan-ru chen (c @yahoo.com.tw) antimicrobial resistance and infection control , (suppl ):p background urinary tract infections (utis) are one of the most common sites of health care-associated infection, accounting for about %, of which about % are related to the use of catheters. in our subacute respiratory care unit, % of the cauti cases were men, whereas those who performed the technique clinically were nurse practitioner, through clinical monitoring and found that nurse practitioner to implement this technology compliance rate of only %. the aim of this study was to assess the feasibility and compliance of catheterization among male patients with competition and questionnaires. the standard operating procedures of "male catheterization" were developed and practiced in clinical practice by means of practical operation and questionnaires. antimicrobial resistance and infection control , (suppl ):p nebulizers are widely used in clinical for treating the disease of respiratory tract, which can aerosolize medicine to . - μm to reaching the lower respiratory tract, so as to induce expectorant action or treat the inflammatory of respiratory tract. however, nebulizers can be contaminated by the exogenous route owing to improper care, so that increase risks of oral colonization or respiratory tract infection. the previous study showed that suggestion about the frequency of replacing nebulizers and nebulizers practices were vary variable and inconsistent. the aim of this study was to evaluate the effect of replacing nebulizers (at , or hours) in contamination. a randomized controlled trial study is conducted from july , to february , in a surgical ward of a medical center in taipei. during the study period, include above years old people with steam in halation treatment. after participants agree to join the trail, randomise cases into , or -hour group. in the protocol, researcher collect culture of nebulizers after used. total specimens of nebulizes are collected. the rate of contaminated nebulizers at hours is . %, which is significantly higher (p = . ) in hours ( . %) and -hour group ( . %). multivariate statistical analysis show that steam inhalation increase by one day with the contaminated risk of nebulizers increased by % ( % confidence interval: . - . , p = . ). the replacing nebulizers more than two days will increase contaminated rates significantly. therefore, health care workers should understand and comply with guidelines of steam inhalation clinical care to reduce the contaminated risk of nebulizer. educational programs increase the compliance of bundle care: experience in a regional hospital in taiwan antimicrobial resistance and infection control , (suppl ):p taipei veterans general hospital yuanshan branch is a bed regional hospital that provides single veterans acute and chronic care. there is a team of care providers helping the nursing staff manage patient's daily care. urinary tract infections (utis) are common hospital-acquired infections accounting for % of hais. recent studies and guidelines suggested that multidisciplinary care bundle could effectively reduce the incidence of catheter-related urinary tract infections (cautis). the aim of this study was to evaluate the impact of educational programs for care providers on compliance of cautis bundles. the intervention study was conducted over a period of years, between and . during a pre-intervention phase, parameters of compliance including the correct rate of had hygiene and correct rate of aseptic techniques were collected and compared with those during the post-intervention phase. the educational programs included ( ) posters reminders on the walls of bedside, ( ) aseptic urine sampling and cleaning technique, ( ) correct draining bag positioning, ( ) frequent tests and feedbacks to supervisors, ( ) hand hygiene. during the study period,a total of care providers received the training programs. the pre-intervention phase was between and , the post-intervention phase was . the correct rate of hand hygiene increased from . % to . %; the correct rate of aseptic techniques increased from . % to . %. our investigation showed that implementing educational programs for care providers increased the compliance of bundle care of cau-tis. however, whether the improvement reduced the incidence of cautis needed further investigations. impact of implementing vap bundle: experience in a veteran's regional hospital in taiwan tzu antimicrobial resistance and infection control , (suppl ):p experience in a veteran's regional hospital is a bed (in intensive care unit is a bed), many critically ill patients need to rely on ventilator for survived. although preventable, ventilator-associated pneumonia (vap) is a main cause of death due to healthcare-related infection in the intensive care unit (icu). recent studies and guidelines suggested that multidisciplinary care bundle could effectively reduce the incidence of catheter-related infections. the aim of this study was to assess implementation of vap bundle, can be effective in preventing ventilator-associated pneumonia occurred. materials and methods vap bundle care was implemented in a medical icu of our hospital from march to march . the contents of vap bundle checklist including daily ( ) readiness-to-wean assessment daily, ( ) diary "sedation vacation", ( ) hand hygiene, ( ) oral care every hours, ( ) head-of-bed elevation above degrees. education, training, and regular feedback from external auditing units can encourage the clinical care team to improve implementation rates. the results showed a daily goal checklist implementation rate increase from % to %. the ventilator bundle compliance rate, assessed by external audit, increased from % to %. the ventilator usage rate decreased . % after implanted vap bundle. the implementation of the vap bundle during year. a record of -month period of zero vap case from march to march had achieved. our experience has shown that the implementation of vap bundle, can be effective in prevention ventilator-associated pneumonia occurred. in addition, staff education and regular feedback from external audit data can improve implementation rates. antimicrobial resistance and infection control , (suppl ):p background ventilator-associated pneumonia (vap) is a major healthcareassociated infection worldwide. the incidence of vap in our hospital was around episodes/ ventilator-days for many years. this study aimed to reduce vap rates among patients in icus. this prospective study was done among mechanically ventilated adult patients in every icu between january st, and december st, . a total of , ventilation days were observed. a element care bundle including daily assessment of readiness for ventilator weaning, hand hygiene, aspiration precautions, prevention of contamination and oral care using . % chlorhexidine mouthwash were introduced to all icu. infection control nurses regularly visited and supervised the practice and periodic meeting among icu nurses were arranged. the adherence was monitored. the incidence of vap was gradually decreased, from . in , to . and . per , ventilator-days in and , respectively. the adherence rate to hand hygiene before tracheal suctioning was . % at the beginning and . % during the last period of the study while adherence to other measures were . % throughout. this study suggests that participation and experience sharing among nurses, regular monitoring, and supervision significantly contributed to the reduction of vap. surveillance of data for post-craniotomy infection were retrieved from infection control unit of songklanagarind hospital. which surveillance system use the former national nosocomial infections surveillance system (nnis) risk index for ssi risk adjustment. the data included patients underwent brain surgeries in the hospital from september to june . medical records were reviewed for additional information including emergency operation, traumatic brain injury, brain cancer, and duration of steroid administration. the predictive performance of the former nnis risk index, new nhsn model, and our proposed model was then compared by mean of area under receiver operating curve (auc). the study identified operations with post craniotomy ssi while the former and new model predicted . and . ssis respectively. auc of the new model ( . %; %c.i = . - . ) was significantly (p-value = . ) less than the former one ( . %; %c.i = . - . ). we analyzed the data and construct a model which included brain cancer, duration of operation more than hours, and contaminated or dirty/infected wound class. auc of our model was . % ( %c.i = . - . ). the new cdc nhsn model is poor for risk stratification postcraniotomy ssi and may be improved by adding brain cancer variable into the model. antimicrobial resistance and infection control , (suppl ):p background catheter-associated urinary tract infections (cautis) are a common infection among elderly in long-term care facilities (ltcfs). nursing personnel play an important role in prevention of cautis. this study aimed to examine the effects of coaching on nurses' knowledge and practice regarding cautis prevention in elderly in ltcfs. a quasi-experimental study was performed during october to january . the participants were nursing personnel working in ltcfs. all samples attended the four steps of coaching intervention including ) preparation; ) discussion; ) active coaching; and ) follow-up. the research tools consisted of a demographic data questionnaire, a knowledge test, an observation form, a coaching plan, and a manual for prevention of cautis. data were analyzed using descriptive statistics, paired t-test, and chi-square test. all of participants were female. the mean age was . years old, and the mean working experience was . years. most participants had no experience in training of cauti prevention in elderly patients ( . %). after coaching, average knowledge scores on prevention of cauti among nursing personnel increased significantly from . points to . points (p < . ) and the proportion of correct practices on prevention of cautis in nursing personnel was statistically higher than the pre-coaching rising from . % to . % (p-value < . ). the findings of this study suggest that coaching could enhance knowledge and practice skills in prevention of cautis among nursing personnel in the ltcfs. application of coaching may improve knowledge and practices in prevention of cautis among nursing personnel in other ltcfs. microbiome-directed therapies are increasingly used preoperatively and postoperatively to improve postoperative outcomes. recently, the effectiveness of probiotics, prebiotics, and synbiotics in reducing postoperative complications (pocs) has been questioned. this systematic review aimed to examine and rank the effectiveness of these therapies on pocs in adult surgical patients. we searched for articles from pubmed, embase, cochrane, web of science, scopus, and cinahl plus. from to , thirty-one articles meeting the inclusion criteria were identified in the literature. risk of bias and heterogeneity were assessed. network meta-analyses (nma) were performed using random-effects modelling to obtain estimates for study outcomes. risk ratios (rrs) and % confidence intervals (cis) were estimated. we then ranked the comparative effects of all regimens with the surface under the cumulative ranking (sucra) probabilities. a total of , patients were included. we found that synbiotic therapy was the best regimen in reducing surgical site infection (ssi) (rr = . ; %ci, . - . ) in adult surgical patients. synbiotic therapy was also the best intervention to reduce pneumonia (rr = . ; %ci, . - . ), sepsis (rr = . ; %ci, . - . ), hospital stay (mean = . days, %ci, . - . ), and duration of antibiotic administration (mean = . days, %ci, . - . ). no regimen significantly reduced mortality. this systematic review suggests that synbiotic therapy is the best regimen to reduce ssi, pneumonia, sepsis, hospital stay, and antibiotic use. surgeons should consider the use of synbiotics as an adjunctive therapy to prevent pocs among adult surgical patients. increasing use of synbiotics may help to reduce the use of antibiotics and multidrug resistance. antimicrobial resistance and infection control , (suppl ):p the incidence of catheter-associated urinary tract infection (cauti) is a major healthcare-associated infection in ramathibodi hospital. the incidence was more than episodes/ urinary catheter-day for many years. we studied the effect of the intervention programs to reduce the incidence of catheter-associated urinary tract infection. a quasi-experimental research was conducted in the surgical intensive care and medical step-down units. the baseline and intervention period was from january to june ; and from september to may , respectively. the cauti bundle according to the cdc's guidelines recommendation was employed. we found non-significant reduction in the rates of cauti ( . versus . episodes/ urinary catheter-day) and urinary catheter utilization ratio ( . versus . ). removal of catheter was not successful in patients with urinary retention, which were major population in our study. multiple logistic regression revealed that duration of hospitalization > days (or . , % ci = . - . ), duration of indwelling catheter > days (or . , % ci = . - . ), and female gender (or . , % ci = . - . ) were associated with a higher incidence of cauti in the medical step-down unit, while > days of hospitalization was associated with cauti (or . , % ci = . - . ) in the surgical intensive care unit. the most common organisms identified in both units were fungus and e. coli. prolonged catheterization is associated with higher incidence of cauti and early removal of catheter may not be achievable in certain group of patients. further study is needed to determine the optimum care of patients who need indwelling catheters. background surgical site infection (ssi) is the most common hospital acquired infection at obstetrics and gynecology hospital. ssi prolonged hospital stay and increased treatment costs. we aimed to identify incidence and causative organism of surgical site infection among cesarean section and gynecology surgery cases. materials and methods a prospective cohort study was conducted at hung vuong hospital, viet nam from january to may . women who underwent caesarean and gynecological surgery were included in the study. cdc criteria ( ) were used to diagnose ssi. results , women underwent caesarean and gynecological surgery. ssi rate was . % ( % ci: . - . %), in which . % ssi after caesarean section ( % ci: . - . %) and . % ssi after gynecological surgery ( . % ssi after hysterectomy with % ci: . - . %). average number of treatment days that with ssi is ± . days. staphylococcus aureus and coagulase-negative staphylococci were the most common organism caused ssi after caesarean section ( %) and escherichia coli was the main organism for ssi after gynecology operation ( . %). the incidence of ssis was limited because we only follow patients with ssis who were treated in hospital. the incidence of ssis after hysterectomy surgery was higher than other types of gynecologic surgery. it's necessary to promote infection control to reduce ssi in hysterectomy surgery. guidelines for the prevention of new strains of influenza infections in employers and employees (update glaxosmithkline biologicals sa. acknowledgements glaxosmithkline biologicals s.a. a total of participants were involved in this competition, with a rate of . %. among them, of them ( . %) were not up to points, and of them were below % according to the technical project contents, respectively: . pre-hand hygiene. . the first disinfection and wait disinfectant dry. . the second disinfection.after the competition, the questionnaire is investigated and analyzed as follows: . professional nurse practitioner years: more than years ( %) accounted for the highest. . % felt it was suitable for clinical practice. . difficult to implement technology: the proportion of the first disinfection accounted for the highest ( %), followed by wearing sterile gloves to no bacteria were connected to the catheter and urine bag ( %), and again the hand hygiene ( %). this technology is standardized and it is recommended to incorporate the new training content of the nurse practitioner. the impact of bundle care intervention reduce catheter-associated urinary tract infections (cautis): experience in a medical center in taiwan key: cord- -esnsa u authors: nan title: abstracts (th) tripartite meeting salzburg/austria, september – , date: journal: langenbecks arch chir doi: . /bf sha: doc_id: cord_uid: esnsa u nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs / year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised dogs with tv, dogs with sgv and dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [ ] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about / year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [ ] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in tv, sgv and pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. and insulin ( . ugkg - ) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was minutes and blood was taken at , , , and then min intervals. gastric acid was measured by autobiuret titration. plasma was stored at - °c until assayed for pp by ria using an antibody sensitive to fmol ml- . results are expressed as mean total increment _+ se. significance: p < . ". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < . ), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < . ). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin ( < . ), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < . ). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than . m nac (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [ ] . we sougth to define the site of this resistance. expt. : gastric emptying of s and mm g was measured in dogs with intestinal cannulas cm distal to the pylorus while gastric pressure was maintained at , or cm h with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. : emptying of s, g, and mm oleate emulsion was measurd in dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at , and cm h . emptying rates expressed as the average of the volumes emptied at the pressures. under both conditions in expt. emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [ ] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in ml meals. gastric volumes were measured by the george technique in dus and n at rain intervals for min after ingestion. gastric ph was maintained constant by intragastric titration ( . for acid; . for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p< . ). with glucose and acid the difference occured within - min whereas with fat the difference occurred between - min. thus ( ) dus do not have a selective loss of inhibition of emptying in response to acid, and ( ) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. gross ra, isenberg ji, hogan d, samloff im ( ) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine mg hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco -transport (flemstroem am j physiol g : ). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for min. ii) normovolemia for min, thereafter min of shock induced by bleeding to mmhg mean arterial blood pressure. iii) min normovolemia and min vasopressin ( . i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = . + . x- . x r= . ; p< . ). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco ( . ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of did not induce oesophagitis. even short periods of oesophagitis ( - weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. patients with typical gastrooesophageal reflux symptoms and control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s- (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = ) the trypsin content was significantly elevated compared to the controls (/ < . wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [ ] . trypsin in gastric juice (ph . - ) will stable and partly active over prolonged periods [ ] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [ ] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [ ] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, %; povidone-iodine, %; chlorhexidine in ethanol, %; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel ( ) devoid of contents ( ) containing bile ( ) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was . the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p< . ) and from one another (/r< . ) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [ ] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b , calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [ ] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = ), ira with a cm reversed loop (ira + rl, n = ) or ira with an artifical valve (ira + v, n = ). body weight, haematological estimations and faecal chemistry were measured weekly for months post operatively. xylose absorption and transit time were measured a minimum of months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e (pge ), on intestinal absorption, electrical activity and volume. in each of dogs a cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at . ml/ rain with a solution of retool/ sodium and retool/ glucose. to measure segment volume, a solution of . g/ mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin . mcg/kg/h ( + . to _+ . ; mean_+sem/ rain) and abolished by (b) intravenous vasopressin . u/kg/h (p< . ). motility was inhibited and mean transit time was prolonged (a) . to . rain and (b) . to . rain (/r< . ). intestinal volume rose by (a) . ___ . ml and (b) . -+ . ml (p< , ). absorption was unchanged by somatostatin and fell with vasopressin ( . --- . to . -+ . ml/ min). acute blood loss sufficient to lower the central venous pressure by cm of water produced identical changes to somatostatin. pge produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was and years respectively. anal canal length was . ___ . cm (mean + s.e.m.) after ia and . ___ . cm after ca. a resting tone of ___ cm water after ia and ----- cm water after cawas recorded. squeeze pressure was ___ cm water after ia and ----- cm water after ca. the recto-anal reflex was present in % of the ia patients and in % of the ca patients. a mean daily bowel frequency of . ___ . followed ia and +_ after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [ ] . lower pressures were found after ca, probably due to the older age of the patients in this group [ ] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in patients with ifi, all of whom leaked during rectal infusion of ml saline, patients with dps, who were continent of rectally infused saline, and matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, - . ___ . cm; dps, - . + . cm) compared with controis (- . __+ . cm;p< . in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, ___ ms; dps, + ms; controls, + ms;/,< . in both cases), and motor unit potential duration (ifi, . ___ . ms; dps, . ___ . ms, control, . + . ms; p< . in both cases). moreover, both groups had an abnormal ano-rectal angle (/,< . ), though this was more obtuse in ifi than dps ( ___ ° vs ___ °;p< . ). however, while patients with ifi had lower sphincter pressures than normal (basal; ___ vs ___ cm water; / < . ; squeeze, ___ vs ___ cm water; / < . ), and required a lower rectal volume to inhibit sphincter tone for more than one minute ( + vs ___ ml; p< . l), these values were normal in patients with dps (basal; ___ cm water; squeeze, _+ cm water; rectal volume, ___ ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [ ] . in the present study using ( c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h -receptor blocker ranitidine ( - molar) on these interactions have been examined. results (percentage of maximal stimulation produced by - molar histamine) results (percentage of maximal stimulation produced by - molar histamine) methionine glutamine alanine amino acids only ( - molar) amino acids and histamine ( - molar) - amino acids and ranitidine ( - molar) histamine and ranitidine -complete inhibition - . the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. . the histamine h -receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. sex related differences in gastric acid secretion have been documented (lilja et al, ) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., ) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. groups of guinea pigs were used for this study, animals as controls, were given estrogens for weeks, were given testosterone for weeks and underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found + . pg/ml, after estrogens . -+ . pg/ml, after testosterone _+ . pg/ ml, after oophorectomy -+ . pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r< . ). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was --+ kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in while patients had greater than grams fat loss per day. weight loss was . _+ . % of recall weight. muscle mass measured by arm muscle circumference ( . _+ . cm) and creatinine heigth index ( ___ %) was normal for our patients but triceps skin fold was % less than normal ( . _+ . mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = ). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was ___ days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= ) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from . cc/min to cc/min. the pancreatic duct was obliterated with neoprene (n= ) silastic (n= ) or left open (n= ). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were . . the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form --+ s.e. pg/ml to over pg/ml following protein meal ( g ground lean beef/kg) in all normal control dogs (n= ), and to levels in the range of to over pg/ml in the same dogs following infusion ofbombesin ( pg/kg/ h) for min. mean basal levels were lower ( ___ . s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising % to % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [ ] , their use in pancreas transplants has not been studied. ~ in patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for days. one patient developed hyperglycaemia coinciding with renal rejection on day . over the previous h pancreas radioactivity had increased by %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining patients had good pancreatic function and no xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was . + . on the third postoperative day and . _+ . at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [ ] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt ) donors and streptozotocin-induced dia- betic da(rt a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july , and march , combined pancreatic and kidney transplantation was performed in patients with type i diabetes who had all been on insulin therapy for at least years. age at the time of transplantation was to years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with to ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the remaining patients initial graft function was excellent with plasma glucose normalizing within hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at to weeks in patients median glucose rose from a basal . mmol/l to . mmol/ and was back in normal range ( . mmol/ ) at h. basal insulin was - pmol/ and also returned to the normal range after h after a peak of - pmol/ at - rain. c-peptide showed a significant peaked rise over basal values ( , pmol/ ) in patients while all values were above pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after and months, respectively. in one of these patients the ogtt after months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at months and an ogtt at sixteen months were normal with insulin peaking at pmol/ and a c-peptide peak of pmol/ at min in the latter. -results at and months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys ( rain in-situ warm ischemia) were auto transplanted after h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a - mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of . rag/ ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of - i.v. daily doses of horse atg alone without additional steroids, on nd- th rejection episodes in recipients of cadaveric renal allografts ( rejections: biopsy-proven) detected within - mos. of transplantation. of rejection episodes, were successfully reversed, with return of serum creatinine to pre-rejection levels in episodes ( patients). three patients had primarily humoral rejections and returned to dialysis - mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all patients during and follwing administration of atg. recurrent rejections ( rd- th) following last treatment with atg ( - mos.) were seen in / patients. all patients had rapid immune eliminiation of atg ( / life - days) as compared to the patients who had no more than rejection episodes ( / life - days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from different transplant centers. patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in groups: ) lew rates were treated with dimethylnitrosamine (dmna), mg/kg b.w., i.v. for acute liver necrosis induction, ) cc ( . ml/ g b.w., i.v.) twice weekly, over weeks for chronic liver damage, ) ccl over weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed and days after dmna treatment and and days after last cc injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared h after removing ( °c) by times perfusion in rain intervals (flow rate ml/g/min with ml/g of ringer). the effect oflp, sera, dmna and cc on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group : , ___ , %, group : , ___ %, group : less than % of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group : ± % group : ___ %, group : nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group . pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic ( ___ % of viable cells) and suppressive for pha response of thymocytes ( _+ % of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [ ] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [ ] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [ ] with specific monoclonal antibodies to lymphocytic helper (okt ) and suppressor (okt ) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in % of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in % of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (< %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g- column passage [ ] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [ ] . comparable percentages of suppressor cells (okt +) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t ), the second tumor was a chemically induced adenocarcinoma of the duodenum (t ). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t was only weakly immunogenic. the doubling time oft i was . days, the doubling-time of the adenocarcinoma was days. intravenous inoculation of isolated t cells led to development of lung nodules which could be counted after days. wag rats were injected i.e. with ml of bn blood or syngeneic blood (controls) at - days before tumor challenge. t l was given in two different ways: a) sc. implantation of+ x mm pieces, b) i.e. injection of isolated tumor cells. t was implanted sc. only. each experimental group consisted of animals. for t it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t lung-metastasis model it was observed that a single bn blood transfusion led to a % reduction of nodules, counted at weeks after inoculation. this reduction in number and size of nodules was highly significant (p< . ). for tumor t , the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at weeks after implantation all tumors in the control group had grown to a diameter of - rnm (average diameter . ram). in the group pretreated with bn blood, only of tumors were palpable at that time (average diamter mm). for t it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by . % polyethylene glycol (peg) and monitored as a od o changes by spectrophotometry. all of the serially studied colorectal cancer patients presented with elevated cic levels (mean = + zt od ) as compared to our standard cic level for pooled normal human sera ( --+_ aod , p< . ). initial values in these patients range from to a od with no correlation to tumor load, site of presentation, or subsequent clinical course. in / patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw ). one ,,antigen" fraction (< . mw) from this complex completely inhibited reference anti-aw binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n-- ) or subcutaneously into wistar-furth rats (n= ) respectively. dfmo ( %) in drinking water was administered to half the animals in each group throughout the experiment. at days ra tumors in dfmo-fed mice weighed % less than tumors in control animals (p< . ); wm tumor weight at days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was . and in ra-bearing control mice was . (p< . ). dfmo caused - % inactivation of tumor odc, reduced ra putrescine levels by /o (p< . ), reduced wm putrescine levels by % (d < . ), reduced wm spermidine levels by % (p< . ) and increased wm spermine levels by % (p< . ). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day the tbw of dfmo-fed mice was . % less than tumor-bearing control mice (p< . ). dfmo-fed mice bearing ra tumors survived . -t- . days longer than control mice (p< . ). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases -ht into the lumen of the feline gut. this study was initiated to: . determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, . to evaluate if this release is under cholinergic or adrenergic control. in cats, cm isolated in situ segments of proximal jejunum were perfused with saline at ml/min ( ~c). perfusate samples were evaluated at rain intervals and concentrations of -ht, sp, motilin were measured by ria's developed in our laboratory. after two -min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically ( v, m s, hz). the output from the loop in ng/ min ( -ht) and pg/ rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at ml min- with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. ; g - ); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of . m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac administration. bombesin ( pmol kg-lmin- ) and neurotensin ( pmol kg-~min ) significantly reduced jejunal water absorption; bombesin and enkephalin ( . nmol kag- rain- ) significantly prolonged tt; and enkephalin encreased water absorption (/ < . in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical -ht responses as above. in additional cats atropine administration (lmg/kg iv) totally abolished the -ht responses to vagal nerve stimulation. the paralled release of -ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in rats (resection of proximal / of small intestine), fasting blood sugars (fbs) decreased , from to mg/dl, and averaged mg/dl at weeks. after ileal exclusion (ie) in rats (resection of distal / of small intestine), fbs fell % in days, from to mg/dl, but increased % above preoperative levels to mg/ dl at weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria ( ml/ h), and glycosuria ( rag/all), were present in ie rats; je rats were normal (urinary output < ml/ h, and urinary glucose mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at , and min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: ( ) normal rats, ( ) stzinduced diabetic rats and ( ) transplanted rats. diabetes was induced by the administration of stz ( mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were ___ , -t- , and __+ pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation ( . . mm) were --+ , ___ , and -+ pg/ min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were -t- , ___ , and + pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/ < . , and diabetes; / < . ). on the other hand, glucagon-stimulated peak values in these groups were _ , ___ , and + pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [ , ] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [ ] with biplanar contrast studies. i at biplanar angiography of ischaemic lower limbs had ais with diameter reduction from - %, of the remainder, were normal (< % stenosis) and were ,,occluded" ('_-- / ). nearly two thirds ( %) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__. / ), upstream abnormality was predicted incorrectly in % and the overall accuracy of clinical examination was %, both for detecting stenosis and predicting its severtiy. aspi (median . ; % confidence ,limits . - . ) and pi ( . ; . - . ) although correlated with stenosis (aspir = . ; p = . , pir = . ; p= . variance analysis for linear regression), did not aid the clinician further (accuracy %). however ltd ( . ; . - . ) was well correlated (r= . , p= . ) and did improve assessment ofiliac stenosis (accuracy %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa mhz, computerized, channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: . (exposed aorta scans); . (transcutaneous carotid scans); and . (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than . mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of patients experience has been gained with the use of eeg, tc m isotope scanning, opg, ct scanning and doppler. in a year follow up over % of patients had a satisfactory outcome. an early mortality of % in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + %. mean volume flow in undiseased arteries ( subjects mean age years) was found to be ± (s.d.) ml/min. from this a lower range for normal flow of ml per minute ( x s.d.) was selected. patients were investigated before surgery and a follow up examination was performed at a mean interval of v months post operatively. groups were defined. group a > ml/min; group b - ml/min; group c < ml/min, of arteries in group a before surgery, remained in the group and dropped to group b. in group b, of arteries, go to group a, remain and dropped to group c. in group c, out of arteries moved to group a and to group b. thus of arteries with below normal volume flow before surgery, were returned to normal range and further improved. remain unchanged and disimproved. of the arteries examined ;/ months after surgery, are in the normal range and a further improved. remain unchanged and disimproved. of the arteries examined / months after surgery, are in the normal range in flow values and a further remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, rats were bled to a mean arterial pressure of mm hg for hrs or % uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding additional rats; controls were only cannulated. twenty pairs were heparinized (h); were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a % survival; % uptake of blood during shock allowed prediction of survival. an additional rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p< . ) was seen in heparinized ( / ; / ) versus nonheparinized rats ( / ; %). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [ ] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = mm hg for h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about %. the enk-antibodies crossreacted with less than %. five dogs received an i.v. bolus of naloxone ( mg/ kg) and a subsequent naloxone infusion of mg/kg/ h after h of hypovolemia. eleven dogs served as control and received equivalent volumes ( mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated - fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [ t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f- has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. < %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f- with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to minutes of hypoxia (fio = , %) and hemorrhagic hypotension (map = mmhc), then randomly allocated to one of three groups and resuscitated by fio = % and infusion of either f- , bsa, or wbs. cytochrome a, a redox state was monitored continuously at run. thirty additional rats were sacrificed at baseline, end shock and and minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as % in the bsa and f- groups, there were no significant differences / < . between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are < % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h (pgh ) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of c-pgh produced only prostacyclin (pgi ) as measured by the formation of its stable breakdown product -keto-pgfla. the amount of pgi formed varied from - % of the substrate depending upon the microsomal protein and pgh concentrations. in addition, the nonenzymatic breakdown of pgh resulted in the formation of pgf a, pge , pgd and heptadecatrienoic acid (hht). there was no formation of thromboxane a (txa ) as measured by the absence of its stable breakdown product txb . in the presence ofgsh, a required cofactor for microsomal pge isomerase activity, the formation of pge was augmented fold (up to % of the substrate) indicating enzymatic for-mation of pge . the gsh either did not alter or augmented (less than fold) the formation of pgi . the increased formation of pge in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh to pgf a, pgd and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, grafts with restricted flow ( cc/min) were removed on the th postoperative day (group i); grafts with unrestricted flow ( ___ cc/min after placement) were taken out days later (group ii) and grafts placed in the j.v.'s were removed days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: . anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi . this suggests that antithrombotic therapy may be necessary to prevent early failures. . huvg develop the capacity to produce pgi and fibrinolytic activators and . although the etiology of nfh remains obscure, the decreased levels of pgi in group ii suggest that exhaustion of pgi generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i s-albu -composite fibrin plate assay size oflytic zones (mmx) after h incubation at °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n= ), ql and clearance increased five-fold (p< . ), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n= ) provoked a slight increase in ql (p< . ), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose ( rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [ ] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coin- before aspirin after aspirin p = [ ] . the experiment was repeated h after each subject had ingested . g of aspirin. the results are summarized in table . . there appears to be a significant increase in granulocyte fibrinolytic activity h after the ingestion of .sg of aspirin. . this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. . aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. . the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel- of hamsters in the event of hemodilution without and in conjunction with chemotherapy ( mg/m dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran . after inoculation of x cells of the amelanotic hamster melanoma a-mel- into s.c. tissue in the preparation, this tumor re~ched a diameter of approx mm within five days. the reduction of systemic hematocrit from . to . ( . ± . ml blood vs dextran , animals) at a tumor diameter of mm increased the growth rate of the melanoma by about % while enhancing significantly the volume flow through capillaries and the mean local po . table control hemodilution capillary velocity (ram/s) . _ . . ± . capillary blood flow (ml/min x - ) . ± . . ± . mean local po (mmhg) . ( - ) . ( - ) the frequency distribution of local po on the tumor's surface showed a distinct shift toward higher po values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. mm for a period of ten days with chemotherapy alone ( animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed ( animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of %. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [ ] physiol rev. : ), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n= ), weight approx. g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx ( mg/kg -oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration h), preceded by a h fasting period were carried out - days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline ( ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next . ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx ( ___ pg/ ml) and elevated in c-sx + amx ( ___ pg/ml) when compared with sham rats ( --. pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx ( . --+ . , . --. . , resp.) when compared with sham ( . --_ . ). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: . stress hyperglucagonemia in the rat is confirmed (levels during zero stress - pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); . the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; . mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. . during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a - % reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following mci mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in control individuals who had :> % left ventricular ej ection fraction and < s pulmonary circulation. thirtythree studies were obtained in patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was s.d. > control mean ( - . ___ . x - units/ min). among positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was . _+ . mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o was . +-- . on . cm h peep, which were both significantly (p< . ) different from patients with normal si's. positive si's were present from hours to days following the apparent onset of ards in patients. recovery of gas exchange was associated with normal si's on repeat studies in patients. of patients with cardiogenic pulmonary edema, had negative studies ( - mmhg pcwp) and i a positive study ( mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at - /min at a fixed temperature of °c measured at the exit of a mm diameter pipe and °c at the site of bleeding. the generator sustains usual steam sterilization. dogs were operated on both patellae, femoral short segments and iliac crests giving different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from cm to meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. . five iliac crests were divided and bleeding measured after thermocoagulation. . the segmental femoral resections were thermocoagulated. . the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo- coagulation was directed to the bleeding surface at a cm distane, sweeping it during to seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of meters. there was a significant decrease of particles around the operating site ( % less, at cm and % less, at meters). in vivo: the bleeding was weighted around % less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [ ] gastroenterology : ). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, . days+ . sem, than that of hepatic bile . + . days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), . _+ . ], than gallbladder bile, (csi, . _+ . ). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [ ] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [ ] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= . /~g ml-lh- ) than in unseeded controls ( . /ag ml-~h- ) (/ < . ). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy ( - % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [ ] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. % of the patients got rid of their concrements within a few days, in . % small remnants remained in the renal pelvis, and in . % ( cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [ ] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in out of experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of cases of es performed for calculi indicated that this was a safe (complications % no deaths) and reliable procedure ( % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [ ] and those aspects of its acitivity which control bile flow [ ] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of . sphincter sc, recorded in animals, ( %) were not associated with duodenal acitivity, whereas . of ( . %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc- ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in . in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [ ] . individual bile salts in the peak min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in patients without pancreatic or hepatic impairment (group c), patients with acute pancreatitis (group ap) and patients with chronic pancreatitis (group cp) all with functioning gallbladders, and patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied subjects divided into groups: group a: acute pancreatitis (n= ). group b: patients undergoing biliary tract surgery (n= ). group c: peptid ulcer patients undergoing gastric surgery (n= ). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = ). group f: control group of healthy subjects (n = ). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in cases ( %). the accr was . + . % (mean___ sd) in group f, control group, and . + . % in group a, acute pancreatitits p< . . accr values below % were found in cases of acute pancreatitis ( %). among those patients whose ap diagnosis was confirmed through surgery the accr was . + . % (mean_+sd), higher than in the rest of the ap patients, . ± . % (p< , ). in groups b,c,d and e (surgical groups) the accr before the operation was . ± . %, . ± . %, . + . % and . + . % (mean___ sd), respectively. after the operation it was: . + . %, . + %, . ± . % and . + . %, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p< . ), but not in the other surgical groups. in patients ( %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: . the accr increase in acute pancreatitis (sensitivity: o/ ) . the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of patients with acute pancreatitis developed a fulminant type. were males and females, mean age years (range - years). all patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with ( ) or without ( ) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was days. due to secondary deterioration a pancreas resection was performed - days later in nine patients. patients with acute fulminant pancreatitis died, a mortality of . %. all patients with the mild type of acute pancreatitis survived, thus the overall mortality was . %. none treated by peritoneal lavage only developed diabetes mellitus, whereas out of surviving patients with an additional pancreas resection had this complication. patients with acute fulminant pancreatitis displayed or more ranson criteria [ ] and six died. however, no less than of those with a mild type of acute pancreatitis fulfilled or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is - % -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between % and %. frequently a causal relationship has been defended. recently, however, any causality has been queried [ ] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n= ) and found a coincidence with a coexisting or prior pancreatitis of . % (n = ). from this a causal relationship cannot be concluded. however, out of these patients (i.e. . %) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (< . %) [ ] . none of these patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in patients. in another patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [ ] . in patients with ap the mean serum calcitonin on admission was ng/ , and the peak level mean lag/ (upper limit of normal ng/ ). calcitonin levels were higher in severe than mild ap, and of patients with levels > ng/ , were objectively graded as severe ap [ ] . in the hypocalcaemic patients, with corrected serum calcium < . mmol/ all recorded calcitonin levels > ng/ and had elevated pth. nine of the patients had significantly elevated pth ('> rig/l) and of these only did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of . gm d - , while dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of . gm dl- . wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p< . ), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'< . ). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, , plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion ( . (submax) or . u/kg-hr. (maximal) iv). at least studies were performed in each dog at each dose. after baseline studies, dogs (group e) were given daily intragastric ethanol ( gm/kg-day). dogs (group a/e) were given e and a ( mg/kgday). after - weeks, secretory studies were repeated. results: all dogs gained weight (x = . kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco output by and %, respectively, in group e, submax secretin, but decreased them by and % in group a/e animals. (p= . vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco output in both groups by - °/c (p= . ) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. fasting patients were given glucagon, m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for min. subsequently it was shown that patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between and rain but the values were sginificantly lower in the pancreatic cancer group (p< . at min: p< . at min: p< . at min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [ ] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt , and respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal ( rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f cardiac allografts survice indefinitely (> days); unmodified lew rats acutely reject such allografts ( -+ days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning > days and retransplanted into normal animals were rejected acutely. adoptive transfer of unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection ( --+ days) in b rats; sensitized slwas somewhat more effective ( _+ day). transfer of x syngeneic peritoneal exudate (pe) cells plus sensitized sl caused acute rejection in % orb recipients ( _ days), the remainder experiencing rejection at c weeks. pe cells harvested from rats injected ip with thioglycollate days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity > %). in vitro, b rat macrophages were abnormal, having only % of capacity of normal macrophages to promote production of interleukin (il ) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection ( -+ days) after transfer of sensitized sl plus semipurified il , thus bypassing the above defect. addition ofll to the t cell (purity > °/ ) equivalent of sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection ( -+ days), while further addition of b lymphocytes (degalan bead adherent fraction, purity > %) or macrophages was uninfluential ( ___ days and ___ days, respectively). transfer of il- alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes ( , transferred concomitantly with il ). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il- production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il- by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt a) to lew (rt ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney days thereaf- ter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above mmol/ in pancreas transplantation, respectively. cyclosporin a mg/kg body weight, dissolved in olive oil, was administered intramuscularly for days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within days and heart grafts within days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. adult mongrel dogs, weighing between and kg, were used as recipients for donor jugular vein segments ( - cm) which had been excised and flushed with cc of plasma protein fraction (ppf) at °c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n= ) received untreated venous allografts without subsequent immunosuppression, group ii (n = ) was the same as group i with minimal immunosuppression (azathioprine . mg/kg/day). in group iii (n= ) the animals were transplanted with venous grafts stored in cc of plasma protein fraction (ppf) containing cy a ( mg/ ) at ° c for hours, immunosuppression was as in group ii. in group v (n= ) the animals received allografts that had been cryopreserved in a % dmso solution at - ° c for - days and then the animals had azathioprine as in group ii. in group v (n= ) venous allografts recipients were treated with systemic cya ( mg/kg/day x weeks, followed by mg/kg/day x weeks) as the only immunosuppression. the patency of the allografts was evaluated at , , , and weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = % patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = %). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = . %) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = %). grafts that remained patent for a initial critical period of - weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day was when a plug or sperm were observed in the vaginal smear. fetuses used were of to days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately rats with transplanted liver fragments were killed , , , and days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: . fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. . hepatocytes began to proliferate about weeks after transplantation. . three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. . when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. of patients with first cadaver transplants treated at our institution between dec. and dec. have had pretransplant sx. at six monts, sx patients had % better kidney survival, but this benefit was lost shortly after year and by and years was % and % worse in sx patients. patient survival for sx and no sx was identical for the first year but was % and % worse by and years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, , patients ages - have received first cadaver transplants after having been tested for reactivity to dncb. nine of dncb negative patients had splenectomy as did of dncb positive patients. kidney survival at year for dncb negative patients without sx was %; for dncb negative with sx, %; for dncb positive without sx, %; for dncb positive with sx, %. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, of dncb negative patients with splenectomy died, primarily of septic complications. since survival of sx patients has been % compared to % in non sx patients (p< . ). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [ ] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a m) [ ] . the aims of the present study were: a) to identify changes in psa and a m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p< . ) reaching a maximum at two months. there was no significant change in the plasma a m concentration over the same period. b) the plasma of consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of months and a maximum of months. grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of /t , producing a % inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, % (/ < . ) for those recipients with a high psa as compared to % for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, patients being steroid-sensitive and steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in of the steroid-sensitive patients, and in of the steroid-resistant patients. the observed one year graft survival rate was . % for the whole group, . % for the patients with steroid-sensitive adcc and . % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz= . ). a high incidence of early graft failure was seen in steroid-resistant adcc patients, . % of grafts being lost in the three months after transplantation, as compared with only of graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence < . , hazard ratio (hr) = . ). interestingly it was found to be most effective against axiallary node recurrence (p< . , hr = . ), reasonably effective against chest wall recurrence (/ < . , hr= . ) but conferred no protection against supraclavicular node recurrence (hr = . ) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died ( out of in wp group; out of in dxt group) % in the wp group and % in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [ ] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [ ] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [ ] as well as with an increased growth rate of inoculated tumors [ ] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n > ) were used in immune response studies. group i: high linoleic acid dietl; group i : low linoleic diet , group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: x leukemia cells were injected i.v. and pieces of mm of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [ , ] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, cysts from patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from . - pmol/ . both sodium and potassium content also varied widely (sodium - pmol/ and potassium - ~umol/ ). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p< . ) and a significant negative correlation with sodium content (/ < . ). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts ( ), predominantly sodium cysts ( ) and mixed cationic cysts ( ). the median dhas concentration of the potassium cysts was pmol/ similar to the levels found in human breast secretions [ ]. in contrast the median concentration of dhas in the predominantly sodium cysts was pmol/ and significantly different (p< . ), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<: . ). six patients had bilateral carcinomata at diagnosis and a further developed a contra-lateral carcinoma during the period of follow-up ( to years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi- cal type. of patients with a particular histological pattern of ilc [ ] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, developed a contralateral carcinome, compared with a further in the remaining patients (p< . ). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the treatment arms via sequential manner. doxorubicin, cyclophosphamide, -fluouracil, and vincristine were given intravenously once every weeks. tamoxifen was prescribed in a dose of mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [ ] . as a result of stenosis reducing flow or by platelet embolisation [ ] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [ ] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from . ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that of the carotid bifurcations showed platelet accumulation on imaging. of the atheromatous ulcers demonstrated by angiography were visualised, but only of stenoses greater than per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm , leriche's syndrome , dissecting aneurysm ), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. ( ) produced endothelial injury and a local increase in shear stress in cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness . --+ . mm) which is an area of low flow velocity ( - ___ cm/s at re ) and shear stress ( -+ dynes/cm ) and not at the flow divider (thickness . ___ . mm, p< . ) which is an area of high flow velocity ( --- cm/s) and shear ( -+ dynes/cm ). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed cm x mm diameter dacron velour infrarenal vp in dogs. an average of x cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = ) and control unseeded (n = ) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of - (for fibrin/platelet thrombi) to + (for confluent endothelial coverage). ae-seeded vp had a score of+ . ___ . (mean___ sd) versus. - . ___ . for controls (p< . ). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of p. this material was tested °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at , , , s and min after reestablishment of the blood flow, ° ) for long term biocompatibility as vascular cylinders ( mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at , and days following implantation. satellite lymph nodes were examined by histology. already s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after days it was completely recovered by a - fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis ( . mm), dermis ( . - . mm) and m. panniculus carneous ( . - . mm), average thickness is indicated. , and day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing ( - photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of , and day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on mm wound strips. specimens contained the superficial . , . and . mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on five months old rabbits which were randomly divided into three groups: in group i, animals served as controls ( animals in sequence of days from the th to the th post-operative day), group ii, animals injected with vit b ( mg/kg of body weight/ h) and group iii, animals injected with a placebo ( animals in sequence of days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [ ] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: . no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. . as far as vitamin b treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. . inversely aponeurosis resistance become significantly greater when measured on the th (p< . ), th (/r< . ) and th (p< . ) post-operative day. in mongrel dogs ( x cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i ( dogs) skin bf was determined from the clearance curve for ~ xenon injected intradermally and measured with a computer-linked gamma camera. in group ii ( dogs) subcutaneous pro was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto were measured before and after the flaps were raised and on postoperative days (pod) , , and . pto were taken at various inspiratoric oxygen levels (f~o ) ranging from % (air) to % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was %, % and % of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p< . ). during pod - bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'< . ). tissue oxygen tension showed a dramatic fall pod , and in the rp flaps for all fio , and for all days the values were lower than the preoperative level (p< . ). one rp developed pod distal necrosis and the pro was then even with a fio of %. the mc flap showed an increased pro on the operative day but at pod the values were slightly lower than the preoperative level, but pod , and the values for all fio were higher than for rp flaps (p< , ). at pod the pro reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro when compared to the rp flap. early postoperative pro in the distal part of the rp flaps is critically low despite of increasing f~o to % and increasing bf. differences in bf and pto may be the biologic factors responsible for the superior healing characteristics of the mc flap. ( ) atp~adp + pi (inorganic phosphate) ( ) pcr + adp~atp the net result ofreaotjoxas and is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a / " bore oxford research systems tmr- spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere > and the nmr index = p/(p~+pcr) was . _+ . (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was < . had nmr index which was significantly elevated above norreals ( . + . p< . ) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of normal whites and normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p< . ). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [ ] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion - h(n)-glucose) were measured before and after infusion of na-dl-./ -hydroxybutyrate (/ oh) to raise levels three-to fourfold in fed (n= ), in fasted (n = ) and in fasted-infected (n= ) animals. in fasted-infected animals before infusion ketosis did not occur (/ oh . ± . mm/ fasted; . ± . fasted-infected) and basal glucose turnover was increased ( . ± . /tm/kg/min fasted; . ± . fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose . ± . ram/ befor; . + . mm/ after). glucose production also fell significantly in the fed ( . ± . /~m/kg/min before; . ± . after) and fasted ( . ± . v. . ± . ) groups but was unaffected by infusion in the fasted-infected group ( . +- . v. . + . ). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [ ] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of . mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at °c for min (with intralipid /a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p< . ) to assess its effect in vivo, volunteers were given , units of subcutaneous heparin h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, . mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of weeks. at least weeks prior to the experiments catheters were placed into the animal's abdominal aorta ( ) and caval vein ( ) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, - . - - g n per day, whereas slightly positive n-balances were observed during the control period ( . +__ . g n/day). muscle glutarnine concentrations declined constantly from . + . mmol/ intracellular water to . - - . by % within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from . + . pmol/min in the control state to . + . during dexamethasone treatment indicating a fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. malnourished patients (p) ( m, f + yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer ' = . +- . x; r= . ;/ < . ). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= . ___ . mg %;p.u. = . ---- . mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase + . %), whereas it acts as a negative ap protein in g.c. patients (mean decrease - / ) (p< . ). moreover malnourished g.c. patients present a reduction of cp values ( - %) which is greater than g.c. patients with albumin > . g% ( - . %); this difference is not statistically significant. cancer patients undergoing palliative (n= ) or radical surgical procedure (n= ) show parallel decrease of preoperative cp ( - %), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of non septic patients (cholecystectomy) were compared with those of septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f - mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc- ) and a lithium buffer system (durrum-pico buffers). conclusions: . this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). . the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [ ] . an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: . in hepatectomized rabbits, mitochondrial phosphorylative activity increased to % of the control and the energy charge level decreased from the normal level of . to . at h after hepatectomy (/ < . ). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r= . , p~ . ). . in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from . to . at h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= . ,/~ . ). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. . in hemorrhagic-shocked rabbit with a mean arterial blood pressure of mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r= . , p< . ). . in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from . to . and the mitochondrial phosphorylative activity was enhanced to % of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically . concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in patients who underwent major surgery such as hepatectomy. these patients were classified into groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below . , group b with transient decrease to . , group c with progressive decrease to below . and group d with terminal decrease to below . . all group a and b patients tolerated the operation well. by contrast, the group c patients showed multiple organ failure with % mortality rate, which involved pulmonary failure ( %), hepatic failure ( %), gastrointestinal bleeding ( %), renal failure ( / ), cerebral failure ( %) and coagulopathy ( %). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. in interferon-treated cells, the '- 'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by '- ' phosphodiester bonds and collectively designated '- 'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the '- 'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first h after partial hepatectomy. partial hepatectomy ( rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals ( rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the '- 'a synthetase activity present in the two first removed lobes was defined as %. we observe a very rapid decrease of enzymatic activity which reaches % already h after partial hepatectomy. the lower level of enzymatic activity ( %) is measured between and h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at h: %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place h after surgery. so well differentiated liver cells have elevated levels of '- 'a synthetase. but, after partial hepatec-tomy, the '- 'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between '- 'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al ( ) johns hopkins med, j, : ). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or h fffsting male wistar rats weighing + g were examined after ph at , , , , and h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of % dextrose for h at the rate of . ml/ g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within h partial hepatectomy caused a decrease in hepatic atp which was maximal at h (from . ___ . to . + . /~ moles/g p< . ); in energy charge (atp + . adp/atp + adp + amp) from . -+ . to . ___ . (p< . ) and increase in phosphorylation potential which was maximal at h (from + to _ p< . ). dna synthesis began at h reaching a peak by h. glucose infusion to ph rats suppressed the decrease of hepatic atp, . ___ . / mole/g at h vs . _ . in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at h vs folds in ffa and folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl , brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a % decrease at h and progressive restoration towards normal values up to h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic ( %) glucose infusion: . the effect of glucose on insulin and glucagon blood levels over h; . the repercussion of the insulin/glucagon modified ratio on dna synthesis; . the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard % ph. a % glucose solution or isotonic salin was infused at a constant rate of . ml/h through a cannula placed in the iliac vein. the rats were sacrified at , , , , and h. ( h) thymidyne ( .. /~ci/mmol) was injected h prior to sacrifice and the liver caudate lobes removed for analysis of ( h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin ( . --+ . ng vs . _+ . ng,/ < . ) and decrease of glucagon ( . -+ . ng vs . + . ng, p< . ), with a major switch of the insulin/glucagon ratio at h after ph (from . to . ). dna synthesis started at h in both series, but was very significantly impaired at h in the glucose infused group ( -+ cpm/mg dna vs + cpm/mg dna, p< . ). infusion of increasing doses of glucagon (from . to mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at . mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: . the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. . if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in , we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of acute bleeding from ruptured esophageal varices in patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was _+ mrnhg and the average duration of tamponade was h. the location of the ruptured v~ices observed by this method was in the lower esophagus within cm of the esophagocardiac junction in % of the cases. the bleeding has been stopped in occasions ( . %) by this method. no significant complications other than atelectasis in patients have been observed. in patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by rain. we have so far used the autosuture apparatus, eea ( mm cartridge) in elective esophageal transection of patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [ ] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. rats with dimenthylnitrosamine-induced cirrhosis were used in this study. of the rats received an end-to-side portacaval shunt, had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf ( . _+ . to . _+ . mls/min/ g-~) and whvp ( . _+ . to . -+ . mmhg). however, if the portal stump was arterialised lbf ( . -+ . to . + . ) and whvp ( . _+ . to . _+ . ) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia , usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin > mg/dl, albumin < . girl, prothrombin time > " s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: . total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; . at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and . similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by %; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by % and of factor viii deficient plasma from infinity to s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [ ] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [ , ] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of , or pg/kg body weight of somatostatin followed by a min infusion of either , or pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of pg/kg body weight followed by an infusion of pg/h/kg body weight)there was a rapid decrease in pp ( . + . to . --+ . mmhg), whvp ( . ___ . to . --- . mmhg) and pvf ( . ___ . to . ___ . ml/min). rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin ( pg) produced a significantly greater reduction in lbf than either or pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone ( ) prostaglandin e (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro ( ) . this study investigates the relationship between er status and pge synthesis in breast cancer cells. pge was measured by radioimmunoassay in primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge values have been corrected for the epithelial cellularity of each tumour. pge x corrected pge = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of - histological sections at x magnification. we conclude that er positive tumour cells synthesize greater amonts pge than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [ ] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in patients with invasive breast cancer treated by mastectomy, tumour er was determined [ ] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between and fmol/mg protein. er levels were higher in postmenopausal patients ( - , median , n= ) than in pre-menopausal patients ( - , median , n= ). for the whole group, optimal prognostic discrimination was achieved with a cut-off fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of fmol/mg protein, whilst in post-menopausal patients the optimum was fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [ ] . it is concluded that: . the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; . in our patients, the optimal cut-off was fmol, which agrees with the level suggested by the british breast group [ ], though it differed between pre-and post-menopausal patients; . optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). patients have now been followed for between months and years. patients initially treated with rt received chemotherapy on failure where possible. of patients receiving rt alone, have developed distant recurrence and have died. of receiving cmf alone have developed distant recurrence and have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of months, as an out-patient procedure, without significant side effects. patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of months before the study commenced. in of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on patients after months showed positive and in of these the disease was progressing, and in one it was stable. of the who were negative, were stable and one progressing. of the patients already on therapy had positive cytology at their initial aspiration - showing progressive disease and being stable. patients whose cytology was initially negative became positive months later and all had disease progression at this time_ patients had negative cytology on or more occasions and in only cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment ( - cm from the incisor teeth). patients and methods: patients were prospectively randomised for treatment by pulsion intubation ( patients) or gastric bypass ( patients). non-resectability was indicated by ( ) tumour lengths greater than cm, ( ) tracheal or bronchial invasion, ( ) disrant dissemination, ( ) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in deaths, a mortality rate of , %. gastric bypass resulted in deaths, a mortality rate of , %. complications: intubation was complicated by respiratory infection ( ), tube migration ( ), respiratory obstruction ( ), oesophageal perforation ( ), bleeding ( ). gastric bypass was complicated by chest infection ( ), pneumothorax ( ), wound infection ( ), subphrenic abscess ( ), anastomotic leak ( ), pulmonary embolism ( ), purulent neck discharge ( ). ability to swallow: palliation of dysphagia was achieved in % of patients following intubation and % of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in % of trials (ids ). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from to bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in dogs. although both prostheses were infected by the standard innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (< to ) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds organisms/ml, such differences in infectibility are clinically significant. ids determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the % level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals ( ); tumorbearing animals ( ), without tumor removal; malnutrltioned animals ( ); tumorbearing animals receiving intravenous hyperalimentation ( ), without tumor removal; animals undergoing tumor removal and colonic operation in one session ( ); animals undergoing the colonic operation two weeks after tumor removal ( ). woundhealing was negatively influenced in an early stage of tumorgrowth (f< . ). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt ( ), devereux et al ( ) and garattini and guaitani ( ) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p< . ). irvin an hunt ( ) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results ( emission m a x ± sem): normal blotchy before reduction . ± . . ----- . after reduction . + . none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion ( , ) because of delay in presentation and diagnosis. in a retrospective study of patients with torsion our salvage rate was %. nineteen percent were misdiagnosed as epididymitis by a junior doctor and % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table ) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the patients with torsion and patients with acute epididymitis were analysed by this program ( table ) . the program correctly diagnosed all cases of epididymitis. of patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing ( beats; - beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration ( , , mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged /min and degenerated into ventricular fibrillation within - rain. in conclusion - min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p< . ) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<: . ). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and , , , , , min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco ) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n= ) acc was under normothermia. in group ii mean t was reduced to °c by the administration of cold potassium cardioplegia immediately after acc and consecutively every min. mph at the end of acc reached . -t- . group i) and . ___ . (group ii) (/ < . ); pmco rose from ___ to _+ mmhg in group i and did not change in group ii. tissue content of atp decreases by % group i) and by °/ (group ii) (f< . ); tissue creatine phosphate fell by % (group i) and by % (group ii) (p< . ). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was . +-- . in group i and . _+ . in grup ii (p< . ). irreversible structural demage assessed by electron microscopy occurred in group i - min after acc and was associated with a mph below . . no such damage was observed in group ii. we conclude: . irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; . during h of acc cold potassium cardioplegia does not completely protect against id when mean t is °c; and . on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at , , and °c respectively. the infusate contained na , k , , , or , ca , or mmol/ , and varying amounts of glucose. measured osmolality varied from to mosmol/kg. the hearts were excised at the end of a rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + . adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at °c, there were no such changes except in hearts perfused with retool/ k and mmol/ ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between and mmol/ k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at °c, it averaged . and . /zmol/g (p< . ) after min of arrest induced by and mmol/l k respectively. ca, or retool/ respectively, significantly increased the atp-decay to . and more than . pmol/g/lo min respectively (/ < . and p< . ). hypothermia of °c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by and mmol/l k without/with ca decreased atp by . / . pmol/g (p< . ) and . / . pmol/g (n.s.) respectively within rain. at °c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased . pmol/g during min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp ___ °c) after coronary perfusion with ml cb ( ___ l___c) containing k, meq/l and d, pg/kg. eight dogs had two hours of mi after perfusion with ml cb containing k, meq/l and d, /zg/kg. six dogs received the same treatment as the previous group except that d was increased to /zg/kg for all four perfusions. baseline studies were repeated after min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group . creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in / dogs defibrillation was not required whereas / dogs with cbk and / with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age weeks). grafts measuring cm in length were placed in animals; were implanted with attention to tissue preservation (live grafts) while received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice , or months later. each months animals with live grafts and with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by months, the live grafts had increased . - - % in length and . ___ % in diameter, while length and diameter of the devitalized grafts showed minimal growth ( . ___ /o and . ___ o/o respectively). during the same period the length of the thoracic aorta increased . ___ % in the live graft group and . ___ % in the devitalized graft group. grafts were lined by endothelium and organized into transmural zones. the thickness of each zone in the live grafts remained static from to months, whereas thickness of the middle and outer zones in the devitalized grafts increased to fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio . ___ : . ----- ). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at °c for h. materials were then minced on paraphine plates and washed with . m cachodylate buffer for h and dehydrated in increasing concentrations of ethanol ( %- %) and intermediate exposure to % uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with . % glutaraldehyde (xcd, pap) than those with % glycerol (hd) and % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with . % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding % due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, patients with tetralogy were corrected. of these, ten patients (ages days to years) had absent pulmonary valve. one patient ( days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of . to . associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi ( tissue and prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc- albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased ( . - . (off) to . - . (on); p< . ) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p< . ), due chiefly to an increase in heart rate ( - to - beats/rain; p< . ), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by % (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx ) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi ) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi , patients undergoing cabg were studied. control patients (no asa) were compared to those receiving or mg. asa as a single dose - hours prior to surgery. production of pgi by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of -keto pgf~a following incubation with um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi (pg/mg tissue) and serum tbx (ng/ml) were (mean -sem): therefore, asa mg or mg did not increase blood loss during cabg. however, asa mg, significantly reduced saphenous vein pgi synthesis (/ < . ) while the lower dose asa mg spared the production of pgi . asa is both doses inhibited tbx (p< . ) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the th or th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~. ). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy- ", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on patients: varicose vein ulcers, atherosclerotic leg ulcers, infected traumatic wounds and infected postoperative wounds. whether in rats or in patients, zy- powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after to ]g days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of male wistar rats, intact rat served as controls. the wounds were dosed with - polypropylene sutures using a single sutur technique. after , and days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to days of healing (breaking energy significantly increased in all tissues from to days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between and days of healing, but was unchanged between and days. the biochemical active zone around the incision line was unchanged - mm on each side from day to . conclusion: while the wound tissue itself continues to gain strength during the days ofheaiing studied, the increase seen after days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with rectus abdominis flaps, based on the superior epigastric artery and based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between month and months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast ( patients), extensive radionecrosis of the chest wall ( case) as part of a primary breast reconstructive procedure ( patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour ( patients). of the patients undergoing primary reconstruction required additional subpectoral prosthesis but in adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of ml per day. scintiscanning after red cell tagging with mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. consecutive surgical patients admitted to our institute from may to july were studied. patients undergoing minor surgical procedures (wound less than cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: . thrombocytopenia is not a typical feature of boomslang coagulopathy. . the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. . despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. . support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. . thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. . although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between /min and /min at beat frequencies of to bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with calves in acute and survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis ( , mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight -year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). . blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. . juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in rats. group i = healthy controls. group ii = aspartate ( ml % sol.) was injected i.p. in rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in rats h before killing, and group iv=in another rats days before killing. group iv =in rats aspartate was administered h before an twice after choledocus ligation, and group vi = in another rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after h, and still more after days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach -tetrazolium reductase - . %, glucose- -phosphatase - . %, alphanaphthylacetat esterase - . %, atp-ase - . %, '-nucleotidase - . %, pas-reaction - . %, mgp-staining - . %. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh -tetrazolium reductase + . %, gsp-ase + . %, esterase + . %, atp-ase + . %, 'n-ase + . %, pas + . %, mgp + . % and lipids - . %. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat - . % and alat - . %. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses ( . , . , . and . ivy-dog-units kg-lmin - ) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [ ] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in . % of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. . resting pressure (rp); . pressure increase after injection of ml saline (lml/s) (pi); . time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in . % of cases, false negatives in % % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/ inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes min, requires no surgical skill, vascular occlusion is less than min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in cases ( hemihepatectomies and resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs ( - weeks) were subjected to shamoperation or % partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs to days after ph. the mean pre-operative total liver blood flow increased from . + . (s.e.m.) ml.g-lmin- to a peak of + % on day . thereafter flow declined towards normal by day . the pre-operative portal component was ___ %; this increased to + . % on day and returned to normal level within to days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm radial compression module with a radial-pak c , ,u, mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of . ml min- with a mobile phase of methanol:water ( : v/v) containing . % (v/v) acetic acid and adjusted to ph . with m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the conjugates alone were completely separated in under rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c cartridge before being applied ( /a to pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml- sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged %. jib patients had progressively greater losses with increasing preoperative weight (/ lbs- %, - lbs- %, - lbs- %, - lbs- %, lbs- %). weight loss in patients dbs was statistically and clinically greater with jib. . diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. . hypercholesterolemic patients had % decreases, from to mg/dl, after jib, but only % decreases after gb. all serum cholesterols were normal after jib, but % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past years jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was %, the postoperative mortality rate %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in . twenty patients with a mean weight of kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. cm of the jejunum and cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels ( . to . ) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in cases and in the left thoracic cavity in cases. functions of stomach placed in the posterior mediastinum were examined in patients surviving more than years and in patients surviving less than years. within one year postoperatively, the absorption ofvitamine b decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all patients surviving over years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in patients surviving years, but extended to the whole surface in patients surviving over years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting . peoples' friendship university, tamojenii pr. , department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from days to years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg ° intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, ). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first or months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to . +__ . , . +--. . (p< . ) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in or months the kidney circulation was restored; in or , and in or months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after or months and remained stable during years (according to the groups . +___ . , . - - . , p> . respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade ) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade ) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade ) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within - h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between ° and °c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance ( - min; °c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after rain and °c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than . (outer stripe of medulla), and medullary lactate levels of to pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above °c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__.. °c; min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys ( to min of warm ischemia; °c) are having polyuric arf. under these conditions, renal ph will not increase above nmol/ h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is rain- °c and seems to be limited by a transitory secondary postischemic oliguria ( - h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within to days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in of pairs of liver allografts, mlc responses were depressed up to fold for weeks post-operatively. in all these pigs, the mlc responses were initially high. in pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after hours incubation at °c in % co with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was %___ % and the viability by trypan blue dye exclusion was at least % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in % of the cases (n= ) the correlation was within cm h . therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [ , ] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n /o . blood was withdrawn to achieve a mean arterial pressure (map) of mmhg (t--- ) which was maintained until t= h when the reservoir was clamped and the animals treated with either nal at mg/kg bolus plus mg/kg.h infusion i.v. or . % nac in equivalent volumes. there were no significant differences between nal (n= ) and con (n= ) in map, left ventricular contractility (lv dp/dt max, mmhg. vs), and survival; the nal animals, however, were acidotic (pha . ----- . ) and colder ( . - . °c) than con ( . +__ . , . -+- . ). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in % of more than mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in % of the dogs. the method of surgical treatment performed were ( ) closure of entry by direct suture, ( ) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and ( ) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in dogs treated by methods ( ) or ( ) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within month and dissection was found to have completely healed on autopsy. in chronic cases treated by methods ( ) or ( ), false lumens were all patent with various degrees of formation of mural thrombi at observation of to months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if ( ) there are substantial doubts about the effectiveness of the intended operation and ( ) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y ql , u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in - h and animals die after ca. h. five male pigs, aged - months, weighing - kg were studied ca. h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x ram) and ionexchange resin (dowex lx ), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially ( , , , rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/ -end, pg/ml, by ria) was related to t:fl-end = (t- ) + , r= . , p< . . when acid-base balance and temperature were maintained, nal (n= ) significantly (*p< . ) improved map and lv dp pg/ml in nal and + pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (> °c lasting more than days) (fuo) and miscellaneous. incidences: patients ( %) had postoperative septic complications. wound infection was recorded in patients ( . %), respiratory tract infection in ( %), urinary tract infection in ( %), fuo in ( . %) thrombophlebitis in ( . %) and miscellaneous infections in ( . o/ ). predisposing factors: wound infections were / ( . %) in clean operations, / ( . %) in potentially contaminated, / ( %) in contaminated and / ( . o/ ) in dirty.wound infections were diagnosed later (mean th day) in clean than in dirty operations (mean th day) (p< . ). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from . % in patients operated on within - days to . % for patients operated after days or more (p< . ). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period ( % vs %). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia ( . %----< min, / > < min, / > min;/~ . ). bacterology: out of cultures gave positive results ( %): aerobes were isolated in samples ( / ); mixed aerobes and anaerobe in ( . o/ ); anaerobes in ( . %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination ( . % in clean operations, . % in potentially contaminated, . % in contaminated and . % in dirty; p< . ).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was days, whereas patients with no postoperative sepsis were discharged after an average of days (p< . ). mean daily cost in our hospital was extimated $ ; accordingly, the mean postoperative hospital stay of patients with sepsis costs $ vs $ of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of nmri-mice ( - g) have been subjected to: sham-operation, splenectomy, peritoneal splenosis, controls as non-operated group.the technique was similar to the one performed in rabbits, prior reported. weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of °c/ml down to c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles weeks postoperatively bacterial challenge with staph.aureus was contradictory: x s c/ml was survived by all splenectomized mice, by only percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [ ] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: . mg, . rag and . mg respectively of crude. d. typus venom were injected subcutaneously in adult baboons. serial determinations of the flow parameters were done over - days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in patients. . two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a ( . - . nmol/mg protein, normal; . ). hepatic insufficiency was observed in patients with cytochrome a contents less than . or more than . . . in all patients with cytochrome a of . - . nmol/mg, the blood glucose level after an oral glucose load ( g) returned toward normal within h (parabolic pattern). in patients with cytochrome a of more than . , the blood glucose level did not return toward normal within h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form . to . .in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at , , , , , and h after sham-operation or % partial hepatectomy (ph). group : carbon suspension (pelikan ink, wagner) mg/ g rat single injection pre-operative; group : olive oil ( % in % dextrose water + . % tween ) . ml/ g rat single injection pre-operative; group : as group given at intervals of h; group : glucan . rag/ g rat pre-op, and at intervals of h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [ ] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from normal weight patients, morbidly obese patients (mean body weight + kg) and patients or more years (mean -+ months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy - mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between and kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was / of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat . %, protein . %, glucosides . % and minerals . %. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at °c± . the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p< . and p< . ). the results of adc expressed in % ± sd were.by roviding a functional gastric capacity of less than ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: . complications occured more often following gastroplasty, . % compared with . % of the patients treated by gastrogastrostomy. . the amount of excess weith lost was greater after gastroplasty, a mean of % for patients followed for two years, than after gastrogastrostomy, a mean of % for patients followed for two years. . because of the incidence of stomal obstruction requiring reoperation following gastroplasty, . % compared with . % after gastrogastrostomy, is our preferred operation. . to improve weight loss after gastrogastrostomy the ta- is now used instead of the ta- stapling instrument. the proximal gastric pouch is reduced in size to - ml and the stoma made smaller to ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of dogs ( %) survived more than months without complications, and of them more than years. in of of the prolonged survivors, extrusion of the graft was recognized in the rd to th month after operation. esophageal stenosis increased slightly up to the th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: dogs weighting - kg were used. these dogs were divided into two groups (control group, n= ; treated group, n= , venalot ® dose: rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of /~m immediately after, as well as days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline ( °c) after the collagenase digestion. the tissue fragments were washed once more and then another mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= ) served as pancreatectomized non-transplanted controls, group ii (n = ) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= ) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine ( rng/kg/day x , followed by , mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of liver allografts and liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res : ( ). the distribution of death among allallograft groups show blocks of survivors. the first block includes animals surviving up to days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord- - ut bu authors: lane, j. michael; summer, lila title: smallpox as a weapon for bioterrorism date: journal: bioterrorism and infectious agents: a new dilemma for the st century doi: . / - - - - _ sha: doc_id: cord_uid: ut bu smallpox, the only disease ever eradicated, is one of the six pathogens considered a serious threat for biological terrorism (henderson et al., ; mahy, ; whitley, ). smallpox has several attributes that make it a potential threat. it can be grown in large amounts. it spreads via the respiratory route. it has a % mortality rate. the potential for an attack using smallpox motivated president bush to call for phased vaccination of a substantial number of american health care and public health workers (grabenstein and winkenwerder, ; stevenson and stolberg, ). following september , , the united states rebuilt its supplies of vaccine and vaccinia immune globulin (vig), expanded the network of laboratories capable of testing for variola virus, and engaged in a broad education campaign to help health care workers and the general public understand the disease (centers for disease control and prevention, a). this chapter summarizes the scientific and theoretical bases for use of smallpox as a bioweapon and options for preparation for defense against it. smallpox, the only disease ever eradicated, is one of the six pathogens considered a serious threat for biological terrorism (henderson et al., ; mahy, ; whitley, ) . smallpox has several attributes that make it a potential threat. it can be grown in large amounts. it spreads via the respiratory route. it has a % mortality rate. the potential for an attack using smallpox motivated president bush to call for phased vaccination of a substantial number of american health care and public health workers (grabenstein and winkenwerder, ; stevenson and stolberg, ) . following september , , the united states rebuilt its supplies of vaccine and vaccinia immune globulin (vig), expanded the network of laboratories capable of testing for variola virus, and engaged in a broad education campaign to help health care workers and the general public understand the disease (centers for disease control and prevention, a) . this chapter summarizes the scientific and theoretical bases for use of smallpox as a bioweapon and options for preparation for defense against it. variola major, the virus that causes smallpox, is an orthopox. variola minor, its less pathogenic cousin, has little theoretical expectation for use as a bioweapon. v. major is a large dna virus ( ϫ nm), with one of the most complex genomes of human viruses ( kbp double-stranded dna). the genome of several strains of v. major has been completely sequenced, but the functions of the genes have not all been elucidated (moss, ) . like most orthopox viruses, variola is host specific. humans are the only natural hosts. experimental infection of small numbers of monkeys with large intravenous doses of virus has been accomplished (leduc and . the virus grows well on many tissue cultures and on the chorioallantoic membrane of embryonated chicken eggs. vaccinia virus, a close cousin of variola, is routinely grown in many laboratories, and can be lyophilized to ensure stability to heat. the same techniques could be used with variola. the genetics of vaccinia are well known, in part because vaccinia strains have been proposed as carrier viruses for genes from other agents, including hiv (moss, ; smith et al., ) . this work shows that the genes of orthopox viruses are amenable to deletions and additions. researchers working with ectromelia (mousepox), another close cousin of variola, have created a very virulent strain, able to escape the protective effect of prior immunization with vaccinia (born et al., ; jackson et al., ) . recent research suggests that smallpox virus could be recreated by synthesizing long strands of dna, thus enhancing its availability for bioterrorism. the viral genome has been deposited in public databases, making such work possible although time-consuming and highly technical (wade, ) . variola virus is fairly hardy in the environment if protected from heat and ultraviolet light. however, it is easy to kill with standard hospital disinfectants or ultraviolet light (fenner et al., ) . there is little information about its ability to survive when aerosolized; by analogy to vaccinia virus, it probably could survive for an hour or more if not in direct sunlight (harper, ; thomas, ) . in summary, variola has several virologic attributes that make it attractive as a terrorist weapon. it is easy to grow. it can be lyophilized to protect it from heat. it can be aerosolized. its genome is large and theoretically amendable to modification. the pathogenesis of smallpox is believed to resemble that of mousepox (ectromelia), which was elucidated by fenner and colleagues during the s and s. subsequent work with rabbitpox and monkeypox, orthopoxes that also cause species-specific systemic disease, have refined our understanding of pathogenesis (fenner et al., ) . infection is via the respiratory route. during the first or days after infection, the virus multiplies in the epithelium of the upper respiratory tract. it is then released into the bloodstream in a primary (asymptomatic) viremia. this viremia is cleared by the reticuloendothelial system, where the virus again multiplies. about or days after the initial infection, the reticuloendothelial cells release a secondary viremia, which is probably cell-associated. this is a massive viremia that causes an intense and prostrating prodrome, with fever, myalgias, and other symptoms of a vigorous viremia. the secondary viremia is cleared toward the end of the prodrome, when the leukocyte-associated virus becomes localized in small blood vessels in the dermis and upper respiratory epithelium. the skin lesions evolve in a stately and characteristic way (see section on "clinical disease"). the histopathology is characteristic. the lesions are full of virus. the fluid from vesicles and pustules and scabs is infectious, and virus can be isolated from scabs (breman and henderson, ; fenner et al., ) . the main source of natural infection is the secretions from the upper respiratory tract, where lesions quickly break down and excrete virus because the epithelium in the nose and throat lacks the firm keratinized layer that seals in the virus in the lesions on the skin. patients are therefore infectious from about - hours before the initial faint macular rash appears on the skin, and remain infectious throughout the course of the rash (breman and henderson, ; dixon, ; fenner et al., ) . the clinical illness and fatality rate roughly parallel the density of the skin lesions. when lesions are sparse, cases are unlikely to die and probably are not efficient transmitters. however, their mobility may allow them to have enough social interaction to result in transmission. vaccine-modified smallpox can be very mild and nonfatal, although if vaccination was more than years prior to exposure, the fatality rate is not trivial and patients can still transmit the disease. as lesions become denser and confluent, the fatality rate increases, the amount of virus in the respiratory secretions increases, and patients are more infectious (fenner et al., ; mack, ; rao, ) . hemorrhagic smallpox has a fatality rate of nearly %, and patients are highly infectious. about - % of unvaccinated patients with v. major get hemorrhagic smallpox, probably with disseminated intravascular coagulation (bray and buller, ; mckenzie et al., ; rao, ) . they are usually very sick, usually unable to get out of bed and thus may not transmit efficiently. the clinical presentation (from mild to discrete to confluent to hemorrhagic) is a function of the host response, not the virus. the clinical types do not breed true, in that transmission from any patient can give rise to any of the clinical presentations, and the virus is the same. the immune response in smallpox includes both cell-mediated immunity and production of neutralizing antibodies. both probably appear about days after the onset of the rash (fenner et al., ) . immunity is essentially life-long in recovered patients, although very rare cases of second infections have been reported several decades after initial infections. the immune response in hemorrhagic smallpox is probably poor, contributing to the very bad outcome. in summary, smallpox causes an acute illness with a devastating prodrome, with virus primarily transmitted via respiratory secretions. the fatality rate roughly parallels the density of the skin lesions and hence the intensity of the preceding viremia. the clinical spectrum of variola major has been well described (breman and henderson, ; dixon, ; fenner et al., ; rao, ) . the illness starts with a dramatic prodrome, with high fever and signs and symptoms indicative of massive viremia. the patient usually improves somewhat when the viremia is cleared, although the fever does not return to normal. as the fever decreases (about - days after the onset of the prodrome), the characteristic rash becomes evident. during the first day or two of the rash, it may be impossible to distinguish from measles or many other viral exanthms. on dark skin, the rash may not be apparent on the first day or two, being simply faint macules. if the mouth is carefully examined, an enanthem can be detected. the lesions of smallpox have a predilection for the cooler parts of the body and are most dense on the face and peripheral extremities. lesions are present on the palms and/or soles in the majority of cases. the individual lesions undergo a slow and predictable evolution. excellent photographs can be found at the centers for disease control and prevention (cdc) website (centers for disease control and prevention, a; world health organization, ) . by about the rd day, the macules become papular, and the papules progress to fluid-filled vesicles by about the th day. these vesicles become large, hard, tense pustules by about the seventh or eighth day. figure . is a typical patient with semiconfluent smallpox on about the th day of rash. the pustules are "in" the skin, not just "on" the skin. they are deep-seated and feel like dry garbanzo beans, usually nearly cm in diameter. about the th or th day, the lesions begin to dry up and umbilicate. by about weeks after the onset of the rash, lesions are scabbing. about weeks after onset, the scabs begin to separate, leaving pitted and depigmented scars. the causes of death from smallpox are not well elucidated. massive viral toxemia probably causes a sepsis cascade. cardiovascular shock may be part of the agonal syndrome. in hemorrhagic cases, disseminated intravascular coagulation probably occurs. antibacterial agents are not helpful. loss of fluid and proteins from the exudative rash probably contribute to death. modern medical care might reduce the fatality rate, but there is no way to prove that contention (bray and buller, ; breman and henderson, ; fenner et al., ; koplan and foster, ; rao, ) . in summary, smallpox produces a serious and prostrating clinical disease. the characteristic pustular rash is easy to diagnose if smallpox is known to be circulating. there is no proven therapy. no data exist to show whether modern supportive care could reduce the death rate. when smallpox is known to be circulating, the clinical presentation and characteristic rash make diagnosis fairly easy. diagnosis can be difficult when smallpox is not high on the index of suspicion. initial cases after a covert bioterrorist attack will probably be missed, at least until the th or th day of the rash. transmission may have already taken place by this time. for that reason, but also as good clinical and public health practice, patients with undiagnosed rashes accompanied by fever should always be isolated until the diagnosis is established. the cdc has produced a diagnostic algorithm based on experience with the differential diagnosis of suspect smallpox cases. this algorithm is figure . and can also be found at the cdc website (centers for disease control and prevention, b; seward et al., ) . most cases initially considered suspect smallpox turn out to be chickenpox, disseminated herpes simplex, secondary syphilis, or drug eruptions. if this algorithm indicates that a patient is high risk to be smallpox, local and national public health authorities should be immediately notified by telephone, and laboratory specimens taken for polymerase chain reaction (pcr), electron photomicroscopy (em), and viral culture. a network of laboratories around the united states can do real-time pcr on very short notice (centers for disease control and prevention, b) . state health department laboratories are or know of the nearest one of these laboratory resource network laboratories. instructions for obtaining, handling, and shipping specimens can be found at the cdc website (centers for disease control and prevention, b) . rapid diagnosis requires a sophisticated viral diagnostic laboratory. rapid testing is best done using em to identify actual virions and real-time pcr assays to detect viral dna. these tests are highly sensitive if adequate specimens are provided to the laboratory ropp et al., ; sofi et al., ) . pcr is very specific, whereas most mammalian orthopoxviruses, including vaccinia and monkeypox, have the same brick-like viral structure and cannot be reliably differentiated from variola with em. public health action should be initiated by either a positive pcr or em test, but confirmation by viral culture should also be attempted. laboratory tests require adequate specimens. copious specimens must be provided if smallpox is seriously suspected (i.e., if the clinical picture fits the "high-risk" category in the algorithm, or if intelligence or communications from terrorists suggests an attack). at least six lesions should be unroofed. the roof tissue and the pus should be placed in separate sterile vials. em grids should be touched to the base of the opened lesions. pus can be dried directly onto plastic slides. punch biopsies of several lesions should be taken, with half put into formalin fixative and half sent unfixed. shipping and handling of specimens are important; guidance should be sought from cdc (centers for disease control and prevention, b). efforts are currently underway to detect smallpox virus in the environment, including in air distribution systems in large buildings. these involve filtration of large volumes of air and testing the material from the filters with pcr (nbc news, ). these techniques are conceptually encouraging, but have unknown sensitivity and specificity. in summary, clinical diagnosis is easy when smallpox is suspected and the rash is fully developed. sophisticated laboratory tests are available to diagnose suspect cases j. michael lane rapidly and accurately. laboratory specimens must be adequate in volume, and carefully handled. suspect smallpox is a public health emergency and proper public health authorities must be immediately informed. there is no nonhuman host for smallpox, and there are no subclinical carriers. once patients have recovered, they are immune and cannot transmit the infection (fenner et al., ) . since there have been no cases since , any new cases must be the result of bioterrorism, or a highly unlikely escape from one of the two official laboratories. the epidemiology of naturally occurring smallpox has been well studied. the work of downie et al. ( ) , rao et al. ( ) , heiner et al. ( ) , mack ( ) , mukherjee et al. ( ) , sommer and foster ( ) , and the recent demographic analysis by gani and leach ( ) provides a good picture of the occurrence and spread of the disease. smallpox does not ordinarily spread rapidly. transmission requires prolonged face-to-face contact, such as that which occurs among family members or caregivers. transmission is most efficient when the index patient is less than feet from the recipient, so that the large-droplet respiratory secretions can be inhaled (downie et al., ; mack, ; sarkar et al., ) . since virus is not secreted from the respiratory tract until the end of the prodrome, patients are usually bedridden when they become infectious and usually do not transmit the disease widely. smallpox has been documented to spread from bedding containing pus and scabs, and from dead bodies (dixon, ; fenner et al., ; hopkins et al., ) . modern hospital systems for handling infectious wastes and cremation of bodies should eliminate such transmission (centers for disease control and prevention, c) . under natural conditions, smallpox is a highly seasonal disease. transmission is most efficient in cool dry seasons, possibly because respiratory droplets evaporate quickly in dry conditions -creating small droplets that remain suspended in the air. although the vast majority of smallpox is acquired by prolonged face-to-face contact, a well-documented outbreak proves that true aerosolization does occur. a patient whose smallpox had not been diagnosed was hospitalized in meschede, germany, in . he was coughing vigorously, which probably contributed to creating an aerosol. patients and visitors on a floor above his room became infected with smallpox (wehrle et al., ) . modern hospital infection control practices should keep such transmission from occurring in hospitals, but the outbreak proves that aerosols can be dangerous. these epidemiological observations led the world health organization in to switch tactics from mass vaccination to the "surveillance and containment" method. this method is also sometimes called "ring vaccination". c.w. dixon describes, in his once-definitive textbook on smallpox ( ), how he used "expanding ring" vaccination to control a smallpox epidemic in north africa after world war ii. vaccine supplies and workers were in short supply, so mass vaccination was impractical. the identification of patients and vaccination of their contacts was his first priority. the next was vaccinating people living in tents surrounding the infected family. finally, an entire infected village was vaccinated if time and vaccine supply permitted (dixon, ) . this concept formed the basis of the surveillance and containment method, after foege and his colleagues made similar observations about the ease of controlling smallpox by vaccinating close contacts in west africa in - (foege et al., foege et al., ) . surveillance and containment methods work well, even in large and geographically extensive outbreaks, such as that which occurred among several tens of millions of people in the ganges floodplain in (fenner et al., ) . it is the method that eradicated smallpox when years of mass vaccination failed. surveillance and containment consists of five steps. the first step is to identify and report cases. the diagnostic algorithm developed by cdc ( figure . ), and the widespread availability of pcr lab tests expedite this process. the second step is to isolate the patient(s). the profound illness and fear of transmission make isolation readily acceptable to the patients and the public. legal authority exists for isolation if necessary (centers for disease control and prevention, c) . the third step is to identify contacts, the persons who might have had prolonged face-to-face interactions with the patients during the time they were clearly ill. often patients themselves are dead or moribund and cannot be interviewed. their family members should be considered contacts, and one or more of them can usually provide information about other possible contacts. contacts are usually easy to find; they want to be vaccinated and will seek health officials once the patient has been diagnosed. the fourth step is to vaccinate the contacts. vaccination prevents smallpox from developing if performed within or days of exposure (massoudi et al., , kennedy et al., . the copious supply of bifurcated needles and lyophilized vaccine now available means that vaccination can be rapid and efficient. the rate-limiting factor will be the paperwork demanded by modern medicolegal systems. the contacts are placed under fever surveillance, with temperatures taken twice a day. if they become febrile, and therefore possibly prodromal, they are immediately isolated before they become capable of transmitting the virus. the fifth step is to vaccinate people who have been or might be associates of one of the first-ring contacts, particularly if the process is not initiated until several days after the index patient(s) became infectious. there is ample time to vaccinate these "second-ring" contacts, because they have not yet been exposed to actual illness. most new cases detected after initiation of containment activities will be known contacts vaccinated in the incubation period and can be promptly isolated. in the unlikely event that a case develops in a missed contact, the containment process is promptly restarted. in summary, under natural conditions, smallpox does not spread rapidly. transmission is by prolonged face-to-face contact. true aerosol spread can occur, but is rare. surveillance and containment tactics should quickly control outbreaks, even large ones resulting from widespread bioterrorist activity. patients suspected of having smallpox must be immediately isolated under full contact and airborne precautions (centers for disease control and prevention, d) . suspect smallpox is a public health emergency, and public health authorities should be immediately notified. smallpox outbreaks in europe and the united states had a high degree of nosocomial transmission (mack, ; mack, ) . if the diagnosis is suspected, modern infection control procedures and effective isolation should virtually eliminate nosocomial transmission. early in a bioterrorist attack, if smallpox is not high on most clinicians' index of suspicion, cases may be missed and transmit the disease before being effectively isolated. most hospitals have procedures to isolate patients with fever and an undiagnosed rash to reduce nosocomial transmission of measles and chickenpox, but these procedures may be difficult to implement in a large, busy city emergency ward. only recently vaccinated personnel should be allowed to attend patients. if the health facility does not have such personnel prevaccinated and designated as clinical team members, anyone entering the room must be vaccinated with fresh vaccine and vigorous technique, and should also wear a properly fitting n- respirator. personnel should be vaccinated even if they claim to have had a recent successful vaccination; if they are immune, such vaccination carries no risk, and it will eliminate the possibility of an error in the vaccination history. supportive care is the basis of the clinical management of smallpox (breman and henderson, ; fenner et al., ) . adequate food and fluids must be provided in a clean environment. if patients are obtunded, intravenous hydration with monitoring of electrolytes is important, although intravenous access may be difficult through the edematous and pock-laden skin. smallpox is disfiguring. older texts suggested removing mirrors from patients' rooms (dixon, ) . there is controversy about the optimal locus for medical care for smallpox patients. there is an ethical imperative to provide the best care possible, but modern hospitals have many immunosuppressed patients (hiv, cancer therapy, transplants, etc.) who would do poorly if they contracted smallpox or had to be vaccinated. facilities with good isolation possibilities -such as motels, older tuberculosis, mental, or veterans administration hospitals, or mobile hospitals, such as those available from the federal emergency management authority or the military -might make adequate smallpox hospitals. medical care can be brought to such facilities, and isolation of the entire facility may be possible. (centers for disease control and prevention, d) dead bodies, bedding, and wastes are potentially infectious (fenner et al., ; hopkins et al., ) . bodies of patients dying from smallpox should be cremated. modern handling of infectious bedding and wastes will kill the variola virus. rooms where patients have been cared for should be disinfected with any standard hospital disinfectant. personnel handling bodies, linen, or wastes must be vaccinated. in summary, careful consideration should be given to the locus of medical care during an outbreak because smallpox is transmissible nosocomially. scrupulous adherence to infection control guidelines must be maintained, including judicious disposal of medical wastes and dead bodies. only recently vaccinated personnel should provide care to patients. no historical evidence exists that smallpox was an effective bioweapon. over several centuries of colonial settlement in north and south america, anecdotes, diaries, and public letters expressed intent to use smallpox against indigenous people. much like the current discussions about the potential use of smallpox as a weapon, what has been written into historical texts and some medical journals may have been fueled more by fear than plausibility. the correspondence between the british general jeffery amherst and his colonels about infecting hostile indians with smallpox is prolifically referenced in recent journals. the general made this suggestion in july , as strategy against tribes near fort pitt, pennsylvania, involved in pontiac's rebellion: "could it not be contrived to send a small pox among the disaffected tribes of indians?" a week later, his colonel replied in the only mention of it ever made again: "i will try to inoculate the ___ with some blankets that may fall in their hands, and take care not to get the disease myself" (knollenberg, ) . several weeks before that communication william trent, an indian trader at fort pitt, suspicious of two delaware indian visitors, wrote in his personal journal, "we gave them two blankets and a handkerchief out of the small pox hospital. i hope it will have the desired effect." intent is clear, but the epidemiological record shows that smallpox was raging among the tribes the previous spring, weeks before these documents were written (knollenberg, ) . more reasonably, the seasonal nature of smallpox caused subsequent outbreaks rather than blankets. the disease, particularly devastating to american-indians, had been endemic among them for over a century. during the american revolution, george washington may have believed that british soldiers infected fleeing citizens by using variolation, despite the fact that variolation was a procedure normally used to prevent smallpox. in an record, a council of indian chiefs faced a pacific fur company trader, who called himself "the smallpox chief," and threatened to uncork a bottle of the virus if the council decided to attack; fear of smallpox may have averted war. oral history relates the use of scabs in blankets, linen, clothing, and virus-contaminated tobacco, spanning activities throughout north america and brazil (knollenberg, ; wheelis, ) . smallpox virus currently exists legally in only two laboratories: the cdc in atlanta and at the state research center for virology and biotechnology in the novosibirsk region of russia. possession of smallpox virus in any place other than these two laboratories is illegal by international convention. a former deputy director of the soviet union's bioweapons program has written that, during the cold war, their laboratories produced smallpox in large amounts, and made efforts to adapt it for loading into intercontinental missiles (alibek, ) . scientists defecting from the former soviet union, or leaving russia seeking work in other nations, may have illegally carried stocks of the virus to "rogue" nations (alibek, ; gellman, ; mangold et al., ; warrick, ) . there is no publicly accessible proof that such defectors actually transported smallpox out of russia, but no way of disproving that they did. allegations have been made that iraq, iran, north korea, and france may have stocks of the virus; these allegations have neither been proved nor disproved (gellman, ; johnson et al., ) . during the middle of , a team of top american scientists found no physical or anecdotal evidence to suggest that iraq was producing smallpox or had stocks in its possession. (linzer, ) . smallpox can be grown in large quantities and lyophilized for stability. large amounts can be stored in relatively small containers. the virus would be relatively stable in an aerosol if protected from heat and ultraviolet light (harper, ) . the infectious dose may be fairly small (fenner et al., ). an outbreak of cases occurred in aralsk, kaza-khstan, in and was kept secret by the soviets. the index case was allegedly on a boat on the aral sea when she was apparently infected (enserink, ; zelicoff, ) a recent claim suggests that an aerosol released from a bioweapons installation on an island in the aral sea infected her while the boat passed close to the island. the fact that only one of several workers on the boat was infected makes this distant mode of infection improbable. the subsequent additional cases, experiencing close face-to-face contact, were consistent with natural spread of the disease (henderson, ) . "dark winter," a widely publicized political exercise intended to educate public health and government leaders about biological terrorism, used smallpox in its script (o'toole and inglesby, ) . the narrative exaggerated transmission rates, and many leaders and media took the fictitious results literally. bbc television followed with a docudrama, "smallpox ," in which a crazed terrorist infected himself and spread smallpox through casual and biologically impossible contact (bbc england, ) . the epidemiology of smallpox renders these fictitious accounts highly improbable. such zeal to alert us to general biosecurity issues obscures many specific aspects of an attack using smallpox. while no official statements identify specific modes of spread that terrorists might choose, unofficial speculations abound. a fear-inducing hoax, or virus sprayed into a building's air circulation system, or the use of nebulizers to infect thousands at a large airport, are all realistic scenarios (bozzette et al., ) . the concept of a volunteer suicidal terrorist who walks around a busy mall, or a big city subway, is unrealistic because smallpox renders people so sick that they are rarely mobile, and they are so visibly sick that they would be avoided by the general public (piller, ) . given the respiratory portal of entry of the virus, a spray or aerosol may be the likely method of introduction. a large aerosol spray from a light airplane, such as a crop duster outfitted to release lyophilized smallpox virus over a public event such as a political rally or sports competition, is technically feasible. the federal aviation administration, or any developed country's aviation security system, would quickly impound such an unauthorized airplane. if smallpox virus is found, the containment efforts, beginning with a public announcement that attendees of the relevant event should get vaccinated, would abort the attack. if smallpox virus is introduced into the air circulation of a large building, large numbers of respiratory infections could take place. several large cities are currently using air filtration systems and doing daily laboratory tests for smallpox, anthrax, and other biohazards. the u.s. government's "biowatch" program deploys devices to sample air throughout cities and operates in open air, but not closed arenas (nbc news, ) . the sensitivity and specificity of these tests in realistic situations have not been determined, but they are theoretically very sensitive because they use real-time pcr. some terrorist groups are associated with the illegal drug trade (hastert, ) . cocaine is sniffed into the upper respiratory tract, so contamination of cocaine with smallpox virus could seed the infection in a widespread population. many drug users do not readily access the health care system when they are ill and might not be quickly diagnosed, and thus transmit the virus. terrorists might spray virus in an airport bus outside the target nation, and infect passengers bound for widespread destinations within that nation. multiple simultaneous cases would thus occur in many cities, with resultant panic. large numbers of smallpox cases are not necessary for an effective bioterrorist attack. a small number of cases could generate public panic. terrorists could put a solution of smallpox virus into hand-held atomizers, and station volunteers outside of such places as entertainment theme parks, military installations, and critical industries. the virus could be sprayed directly into the faces of persons leaving such facilities, under the guise of marketing a new perfume, etc. half a dozen cases, with obvious connections to well-known institutions, could invoke panic and social disruption, such as that which followed the anthrax cases in the united states in . terrorists with access to a modern virus laboratory might genetically modify smallpox in ways similar to the published manipulations of ectromelia (jackson et al., ; roos, ) . such a strain could not be tested for pathogenicity because there is no animal model for human infection. genetically altered strains might pose problems of transmission; alteration of pathogenicity might have unknown effects on the transmissibility of the virus. experienced intelligence observers feel that terrorists would avoid creating a strain with enhanced virulence. such strains could devastate developing countries with poor public health systems, and a widespread outbreak would quickly spread to such countries (johnson et al., ) . natural smallpox could similarly boomerang. terrorists with the ability to manufacture it would realize that an effective attack might cause widespread disease in nations harboring their colleagues. many such nations have poor public health systems and little vaccine, and would be more devastated than the nation initially attacked (johnson et al., ; oxford, ) . in summary, smallpox virus may exist in "rogue" nations. scenarios involving aerosol spread of smallpox are technically feasible, but have limitations. even small numbers of cases might suffice to create considerable panic. the most important aspect of prevention of a bioterrorist attack using smallpox is reliable intelligence. the location of the virus and the abilities and intent of its possessors drive preventive efforts. allegations that iran has weaponized smallpox must be viewed through our knowledge that these allegations have been made by an iranian opposition group (warwick, ) . the claims that france, iraq, north korea, and yemen have the virus await firm data (gellman, ) . unproven statements in an internationally politicized climate must be weighed with evidence-based intelligence. reliable intelligence, the key factor, is an unknown. public health efforts, and the extensive publicity about them, have direct preventive value for averting an attack or a resulting major epidemic of smallpox, and can be emulated in many nations. the announced british strategy is to stockpile vaccine and train a cohort of health workers who can identify and isolate smallpox cases and start surveillance and containment activities (oxford, ) . in the months after september , , the united states increased its vaccine supply from million doses to about million doses. the supply of vig increased more than -fold. the number of laboratories capable of doing real-time pcr for orthopoxviruses increased from two to at least . courses for clinicians, public health officials, and others have been widely held. several new websites have been created to assist professionals with the diagnosis and management of smallpox, and acquaint them with all aspects of vaccination. posters and training materials have been widely distributed. nearly , front-line medical personnel have been vaccinated (centers for disease control and prevention, f) . in the absence of solid information about the risk of a bioterrorist attack using smallpox, nations face a policy dilemma. if vaccination carried no risk, there would be no dilemma; widespread vaccination would be reinstated. however, vaccinia is a pathogenic live virus. vaccination carries well-known risks, particularly in populations that are largely naive to the virus, and in which large numbers of persons are immunosuppressed by hiv, posttransplant therapy, cancer chemotherapy, and steroid medication (lane and goldstein, ) . the potentially fatal complications of vaccination have recently been reviewed (centers for disease control and prevention, e; lane and goldstein, ) . postvaccinial encephalitis occurs about four or five times per million vaccinees, but carries a % fatality rate. progressive vaccinia is rare, but in our highly immunosuppressed population could be more common than in the past. it may be fatal in % or more of cases. eczema vaccinatum is more common, and the background prevalence of eczema (atopic dermatitis) is about three times higher today than in the s when studies of the frequency of eczema vaccinatum were performed. it has a fatality rate of about %. figure . is the face of an eczematous woman who acquired vaccinia from her child. recently, myocarditis has been found to occur about once in every in , vaccinees (arness et al., ; eckart et al., ; halsell et al., ) . postvaccinial myocarditis has an unknown fatality rate, but deaths have been reported in the past (dalgaard, ; ferry, ; finlay-jones, ) . in addition to these serious complications, a number of vaccinees suffer from "robust" major reactions, with fever, pain, and inflammation at the site; about % develop inconsequential, but sometimes, unsightly rashes (frey et al., ; grabenstein and winkenwerder, ) . vaccinia is transmissible by direct contact. transmission is rare and generally only to very close contacts such as family members sharing a bed (grabenstein and winkenwerder, ; neff et al., ) . the presence of immunosuppressed patients on the wards of major hospitals has kept many medical personnel from accepting vaccination. careful management of the vaccination site and scrupulous hand hygiene can minimize transmission . vaccination is not the only means of preventing smallpox. rigorous isolation of patients coupled with quarantine of contacts until their incubation period is over will prevent transmission (bozzette et al., ; eichner, ; eubank et al., ; mack, ; meltzer et al., ) . the prolonged incubation period ( days) provides time to allow public health action and education of the population once initial cases are recognized. in the absence of circulating smallpox, but the presence of a theoretical threat of a bioterrorist attack, there are several options for the use of smallpox vaccine. in , the united states government's advisory committee on immunization practices (acip) recommended that vaccinia be reserved for laboratory personnel who work with orthopoxviruses capable of infecting humans (e.g., variola, vaccinia, and monkeypox) (centers for disease control and prevention, ) . these guidelines were expanded to include animal handlers working with animals infected with orthopoxviruses. after september , , when smallpox vaccine in the united states was limited to million doses, the acip formed a smallpox working group, which held public hearings and studied scientific data and political opinions. many public health and academic officials suggested that vaccine be made available to limited numbers of workers who would form response teams in the event of a terrorist attack. in june , the acip recommended that vaccine be made available to selected state and local officials with responsibility for protection of public health (centers for disease control and prevention d). this was consistent with cdc's plan for containment of a smallpox attack and coincided with proof that the million doses of vaccine could be diluted : (centers for disease control and prevention, d; frey et al., ) , that additional vaccine was on hand, and that production of new vaccine was well under way. vaccine supplies are now more than adequate to immunize the entire u.s. population, and suggestions have been made to resume vaccination more widely (bicknell, ; derugy and pena, ) . public announcements suggested vaccinating considerable numbers of health care workers, particularly in big city emergency wards and other areas where initially undiagnosed smallpox cases might seek medical care. many states sought to revaccinate predominantly older workers who had a past history of vaccination, because the residual immunity in such revaccinees is not trivial, and they are less likely to experience adverse events after vaccination (hammarlund et al., ) . the number of health care workers in big city hospitals is large, and they turn over rapidly. many health care workers vaccinated under such a policy are in frequent contact with patients with hiv, status postorgan transplants, atopic dermatitis, etc. given the recent data (frey et al., ) that adults given primary vaccination frequently need to take one or more days off work because of fever and malaise, many hospitals expressed concern about the effects of widespread vaccination (connolly, ; gettleman, ) . other "first responders" -including policemen, firemen, ambulance workers, and other emergency medical personnel -should be considered for preevent vaccination (bush, ; centers for disease control and prevention, g; stevenson and altman, ) . in part reflecting these concerns, in august the institute of medicine recommended shifting emphasis away from vaccinations to focus on measures to improve coordination and quicken response time to any public health threat (olson, ) . several writers suggest that individuals have the right to decide for themselves about the risks and benefits of vaccination, particularly given the current copious vaccine supply (bicknell, ; derugy and pena, ) . the morbidity and mortality from vaccinia, the problems of screening potential vaccinees and their household members for contraindications, and the efficacy of surveillance and containment methods for controlling smallpox outbreaks, argue against such a policy (bozzette et al., ; eichner, ; eubank et al., ; lane and goldstein, ; meltzer et al., ) . the united states stopped routine vaccination in . it could be resumed if the threat of smallpox becomes considerable. only in a scenario where smallpox becomes widespread would it be wise to resume mass vaccination. such a scenario would inevitably include inadvertent exportation of the disease to other nations, including those with poor fiscal and public health resources and no vaccine. such resumption of endemicity of smallpox would be a human tragedy of untold proportions. thus, developed nations must stockpile vaccine and maintain preventive public health efforts for rapid containment of terrorist-induced outbreaks. in summary, intelligence about the existence of smallpox in the hands of terrorist groups, and their intent for it use, is the best means of preventing an attack. readiness to control an outbreak resulting from an attack entails a high index of suspicion among clinicians, a good network of diagnostic laboratory capabilities, and a plan for use of surveillance and isolation techniques to quickly contain outbreaks. vaccination policies need periodic review, because the risks and benefits of vaccination of various medical and law enforcement groups are controversial. resumption of widespread vaccination is dangerous and unnecessary. the united states has rebuilt its vaccine supply, trained diagnostic laboratories, enhanced the availability of clinical and epidemiologic information, and completed a detailed plan for handling a smallpox attack. what should be done to reduce the threat of smallpox further? there are scientific and political efforts currently underway to prepare for an outbreak, to reduce its likelihood, and to enhance the safety of vaccines. the intelligence community and the new department of homeland security have increased the antiterrorism budget and the amount of attention paid to possible bioterrorist threats. presumably much of this work is classified, but rumors of nations that may still have (illegal) stocks of variola virus are under investigation. rapid detection of variola virus in the environment would be useful. air filtration systems are being improved so that huge volumes of air inside large buildings, such as commercial or government offices, can be routinely and rapidly filtered. the filters are then tested by pcr for the presence of variola virus dna (fackelmann, ; nbc news, ) . the sensitivity and specificity of such systems cannot be calculated, because no real smallpox has been found or can be injected into these systems. given the high sensitivity of pcr, they should be good at detecting variola. a test that could find virus in the throats of patients early in the prodrome could help identify patients who need immediate isolation in the event of an outbreak. such tests have been developed, again using pcr techniques, and are being explored using material taken from experimentally infected monkeys . antiviral compounds, particularly ones derived from cidofovir and its analogues, are being investigated. a nontoxic antiviral with good activity against variola, or indeed against the serious complications of vaccinia, would change the risk-benefit calculations for both pre-and postevent vaccination programs. compounds that are more soluble and thus more bioavailable than cidofovir are currently being screened for activity against variola (bray and roy, ; langbein, ; nyets and declercq, ; smee and sidwell, ) . a vaccine that provides protection against variola but is less pathogenic than vaccinia would be useful. several candidate strains derived from vaccinia are being actively investigated. modified vaccinia ankara, nyvac, and lc m are prominent among these attenuated strains (bonnilla-guerrero and poland, ; drexler et al., ; earl et al., ; henderson, borio, and lane, ; lane and goldstein, ; mccurdy et al., ) . ultimately, the united states, russia, and the world health organization will have to decide whether retaining the existing legal stocks of variola virus is worthwhile. as long as the virus exists in any laboratory, terrorist groups may claim that one motivation for retaining the virus is its potential as a bioweapon. if the stocks are destroyed, this argument no longer exists. international cooperation under the aegis of the world health organization is needed to affirm the illegality of biowarfare using variola, and maximize efforts to find and destroy illegal stockpiles (breman et al., ) . biohazard myopericarditis following smallpox vaccination produced by simon chinn, in association with the learning channel and granada international the case for voluntary smallpox vaccination smallpox vaccines: current and future a poxvirus protein that binds to and inactivates il- and inhibits nk cell response a model for a smallpox-vaccination policy looking back at smallpox antiviral prophylaxis of smallpox preventing the return of smallpox diagnosis and management of smallpox u.s. to vaccinate , workers against smallpox. the new york times protecting americans: smallpox vaccination program smallpox vaccination and myopericarditis: a clinical review smallpox vaccine: recommendations of the immunization practices advisory committee (acip) smallpox images diagnostic algorithm for rash illnesses smallpox response plan and guidelines. guide c. legal and quarantine authority smallpox response plan and guidelines (version . ) guide d-specimen collection and transport smallpox response plan and guidelines (version . ) environmental control of smallpox virus annex . medical management of smallpox patients and vaccination complications smallpox vaccination and adverse events. guidelines for clinicians smallpox vaccination program status by state recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) medical professionals cite possible side effects, uncertainty of threat. the washington post lack of vaccinia viremia after smallpox vaccination fatal myocarditis following smallpox vaccination orthopoxviruses responding to the threat of smallpox bioterrorism smallpox in tripolitania, ; 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mousepox exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox terrorists' most likely weapon here? induction of human t cell-mediated immune responses after primary and secondary smallpox vaccination general amherst and germ warfare. mississippi valley smallpox: clinical types, causes of death, and treatment smallpox and pan-orthopox virus detection by real-time '-minor groove binder taqman assays on the roche lightcycler and the cepheid smart cycler platforms evaluation of ist century risks of smallpox vaccination and policy options transmission of vaccinia virus, and rationale for measures for prevention new compounds show promise as smallpox drugs. reuters strenthening national preparedness for smallpox; an update modelling responses to a smallpox epidemic taking into account uncertainty u.s.team in iraq finds no smallpox smallpox in europe, - a different view of smallpox and vaccination epidemiology of smallpox in west pakistan i: acquired immunity and the distribution of disease an overview on the use of a viral pathogen as a bioterrorism agent: why smallpox? plague war, pbs videotape modified vaccinia ankara: potential as an alternative smallpox vaccine haemorrhagic smallpox. . specific bleeding and coagulation studies modeling potential responses to smallpox as a bioterrorist weapon poxviridae: the viruses and their replication pattern of intrafamilial transmission of smallpox in calcutta government monitoring philadelphia air for biotoxins. sensors check for anthrax, smallpox, in cities contact vaccinia-transmission of vaccinia from smallpox vaccination therapy and short-term prophylaxis of poxvirus infections: historical background and perspective panel urges shift of focus in preparing for smallpox. the new york times shining light on dark winter smallpox bioterrorism unlikely: populations are easily protected. basic notes smallpox strike called unlikely. the los angeles times epidemiological studies of smallpox. a study of intrafamilial transmission in a series of infected families. ind smallpox. the kothari book depot scientists research antidotes to super mousepox virus pcr strategy for identification and differentiation of smallpox and other orthopoxviruses virus excretion in smallpox. . excretion in the throats of household contacts mouse models for studying orthopoxvirus respiratory infection development and experience with an algorithm to evaluate suspected smallpox cases in the united states a review of compounds exhibiting anti-orthopoxvirus activity in animal models the formation and function of extracellular enveloped vaccinia virus real-time pcr assay to detect smallpox virus the smallpox outbreak in khulna municipality, bangladesh. . methodology and epidemiologic findings bush lays out plan on smallpox shots; military is first. the new york times smallpox shots will start soon under bush plan. the new york times vaccination success rate and reaction profile with diluted and undiluted smallpox vaccine: a randomized controlled trial air sampling of smallpox virus mass smallpox vaccination and cardiac deaths us paper to face russian lawsuit. gazeta, ru an airborne outbreak of smallpox in a german hospital and its significance with respect to other outbreaks in biological warfare before an epidemiological analysis of the smallpox outbreak in aralsk key: cord- - qwbkg o authors: hoddle, mark s. title: biological control of vertebrate pests date: - - journal: handbook of biological control doi: . /b - - / - sha: doc_id: cord_uid: qwbkg o nan several species of vertebrates, especially mammals, have been successful invaders and colonizers of new territories, in particular, insular island ecosystems. others have exhibited high environmental tolerance and adaptability after careful and repeated introductions to new locales by humans. following establishment, several vertebrate species have become important pests. these pests harm agricultural systems by damaging agricultural lands [e.g., rabbits (oryctolagus cuniculus linnaeus) in australia and new zealand], by attacking crops [e.g., european starlings (sturnus vulgaris linnaeus) in the united states], and by acting as sources for communicable diseases [e.g., brushtail possums, (trichosurus vulpecula kerr) , are reservoirs for bovine tuberculosis (mycobacterium bovis karlson and lessel) in new zealand]. other pest vertebrates damage natural systems by threatening the continued existence of endangered flora [e.g., goats (capra hircus linnaeus) on the galapagos islands] and fauna [e.g., brown tree snake (boiga irregularis { merrem }) on guam], and by adversely affecting wilderness areas by changing ecosystem functions and diversity (vitousek et al., ) . the continued relocation of vertebrates exacerbates the ongoing problem of global homogenization of biota (lodge, ) . movement of particular vertebrates into areas where they had not previously existed has, in some instances, occurred naturally without human intervention [e.g., the passerine bird (zosterops lateralis { latham }) arrived in new zealand unassisted from australia]. the vast majority of vertebrate translocations have been human assisted. accidental introduction has occurred as a consequence of human transportation [e.g., brown tree snakes, mice (mus musculus lin-naeus) , and rats such as rattus rattus linnaeus and r. norvegicus berkenhout) ]. some releases have been intentional (but illegal) to serve self-centered private interests [e.g., monk parakeets (myiopsitta monachus { boddaert }) in new york, florida, and texas]. other species have been legitimately introduced to procure public benefit by providing: ( ) new agricultural products [e.g., european wild boars (sus scrofa linnaeus); sheep (ovis aries linnaeus); cows (bos taurus linnaeus), goats and rabbits for meat, and brushtail possums for fur in new zealand], ( ) recreation [e.g., red deer (cervus elaphus linnaeus), fallow deer (dama dama linnaeus), and trout (salmo trutta linnaeus and s. gairdneri richardson) in new zealand]; ( ) companionship [e.g., cats (felis domesticus linnaeus) and dogs (canis familiaris linnaeus)]; or ( ) biological control agents [e.g., the european fox (vulpes vulpes linnaeus), stoats (mustela erminea linnaeus), weasels (m. nivalis linnaeus) , and ferrets (m. putorius furo linnaeus) for the control of rabbits in australia or new zealand; cane toads chemical and cultural control of vertebrate pests is expensive and nonsustainable, and at best provides a temporary local solution to problems (hone, ; williams & moore, ) . biological control of vertebrates, a potentially less expensive and self-sustaining method of population suppression, has focused primarily on mammalian pests. predators, parasites, and pathogens specific to mammals with two notable exceptions (the myxoma and calici viruses that infect rabbits) have failed to provide satisfac-tory control (shelford, ; howard, ; davis et al., ; wood, ; smith & remington, ; whisson, ) . historical records indicate that the majority of attempts at vertebrate biological control have been ad hoc efforts and not the product of careful studies designed to elucidate factors and conditions likely to affect the impact of natural enemy introductions on pest populations. furthermore, failure of biological control of vertebrates by predatory vertebrates has compounded problems associated with exotic vertebrates because control attempts result in addition of new species that cause biological and conservation problems. the level of control achieved by natural enemies is dependent on ratios of natality to mortality of control agents and their host species (davis et al., ) . for vertebrates these ratios are affected by many factors: advanced learning; social, territorial, and breeding behaviors; chemical, physical, and immunological defenses; temporal and spatial escape strategies; and genetic selection in both natural enemy and host populations for persistent coexistence. these complex interrelated factors, coupled with opportunistic feeding habits, have made vertebrate pests difficult targets for biological control with natural enemies. advances in understanding of mammalian fertilization biology have provided molecular biologists with necessary information to develop and investigate the concept of immunocontraception for vertebrate pest control. immunocontraception utilizes genetically modified pathogens that express surface proteins from the target pest's egg or sperm to induce an immune response in the host. antibodies then attack gametes in the host's reproductive tract causing sterilization (tyndale-biscoe, b . computer models indicate that immunocontraception may provide long-term control of vertebrate pests because genetically modified pathogens reduce net reproductive rates without killing hosts (barlow, (barlow, , . in this chapter, i discuss attributes that have aided vertebrate establishment; damage resulting from colonization and uncontrolled population growth; biological control of mammalian pest species with predators, parasites, and pathogens; and future directions that biological control research for vertebrates is taking with genetically engineered microorganisms. permissive to successful introductions of new species. e ton's ( ) predictions have been substantiated in part by paleobiological reconstructions of invasions between newly joined communities (vermeiji, ) and by mathematical modeling describing multi-species interactions in communities (macarthur, ; case, ) . isolated oceanic islands (e.g., new zealand), and insular continents and habitats (e.g., australia and lakes) often have a low diversity of native species. such environments have typically experienced little immigration and are susceptible to invasion by vertebrates (brown, ) . stable, speciose communities with high levels of interspecific competition appear to resist invasion by new species and are sources of successful colonists into less speciose or disturbed communities (macarthur, ; brown, ; lodge ) . this phenomenon has produced asymmetrical patterns of colonization, with successful vertebrate invaders usually being native to continents or extensive nonisolated habitats within continents with more diverse biotas (brown, ; vermeiji, ) . continental herbivores and predators have been very successful in establishing self-sustaining populations in insular habitats, in part because such habitats often lack large generalist vertebrates and essentially have just two trophic levels, producers and decomposers (if specialist herbivores and their predators are excluded) (vitousek, ) . niches equivalent to those on the mainland are largely unoccupied (brown, ) . insular ecosystems therefore often appear to readily accommodate generalist herbivores and predators, perhaps because of low levels of competition for resources that are often inadequately defended chemically or physically (vitousek, ; vermeij, ; bowen & van vuren, ) . organisms from insular habitats that have not coevolved closely with predators or herbivores lack life history features that deter attack or permit survival despite high mortality from predation or herbivory (bowen & van vuren, ) . lack of such biological attributes may increase the competitive advantage of exotics (case, ; coblentz, ; vitousek, ) . introduced vertebrates can also be extremely disruptive in continental regions when habitat disturbance by urbanization or agriculture occurs. european wild boars have detrimental effects on gray beech forests in the great smoky mountains in the southeast united states. habitat disturbance-through pig rooting, trampling, and browsing-and human removal of predators (pumas and wolves) aided pig establishment and spread in this area (bratton, ; singer et al., ; vitousek, ) . communities vary in their ability to accommodate the establishment and proliferation of new species (primack, ) . elton ( ) suggested that species-poor communities (e.g., islands) or highly disturbed habitats are more although there are well-recognized exceptions to general rules that characterize successful vertebrate colonists (ehrlick, ; williamson & fitter, b) , species that establish self-sustaining populations outside their native range typically exhibit some of the following general characters: ( ) short generation times, ( ) high dispersal rates, ( ) high tolerance for varying geographic and climatic conditions, ( )polyphagy, ( ) low attack rates from upper trophic level organisms, and ( ) human commensalism (ehrlich, (ehrlich, , lodge, ; williamson & fitter, a) . the assumption of a high intrinsic rate of increase is generally unnecessary for establishment (ehrlich, ; lodge, ) , although it is important for the establishment of some exotic bird species (veltman et al., ) . the spread of the european rabbit in australia and new zealand, countries that both historically lacked significant eutherian mammal fauna, illustrates some of the preceding points. the european rabbit originated in spain and portugal (corbet, ) and spread through most of europe over years ago following deforestation by humans for agriculture and overgrazing by livestock. animals reared in captivity were carried through europe in advance of naturally spreading populations (flux, ) . the spread of the european rabbit throughout australia following its introduction in varied from km/year to approximately km/year. rate of spread was fastest across dry savannas where conditions were most similar to mediterranean climates and slowest through woodlands (myers et al., ) . in australia, habitat alteration by humans such as conversion of land to pasture, overgrazing of rangelands, and predator eradication [e.g., dingos (canis familiaris dingo meyer)] aided rabbit survivorship and spread. rabbits are polyphagous and feed on grasses and browse shrubs. during severe food and water shortages, bark, fallen leaves, seed pods, tree roots, and termites are consumed (myers et al., ) . rabbits are highly fecund and exhibit rapid population growth under good conditions. individual female rabbits to months of age can produce litters averaging to young, and with abundant food to offspring per year are produced (gibb & williams, ) . behavioral adaptability, sociality, and territoriality, in addition to use of elaborate underground warrens, have also aided rabbit proliferation in marginal habitats (myers et al., ) . assistance in establishment some of the first introductions of exotic vertebrates were those commensal with humans as they colonized new areas. among well-documented early human introductions are dingoes in australia (brown, ) and polynesian rats [rattus exulans (peale)] on pacific islands (roberts et al., ) . as early europeans explored the planet, other commensal species such as r. rattus, r. norvegicus, and mus musculus expanded their geographic range without deliberate human assistance and are now cosmopolitan in distribution (brown, ) . not all vertebrate pests cohabitat with or are associated with human disturbance of the environment. red deer and feral cats, for example, inhabit much of new zealand's pristine habitats with no human management. most vertebrate translocations fail even with human assistance. failure of exotics to establish may depend on life history parameters, responses to abiotic factors, inability to outcompete native species for resources or enemy free space, or chance (cornell & hawkins, ) . deliberate releases of exotic birds have establishment rates of around to % (veltman et al., ) . establishment estimates for intentional vertebrate releases are biased because successes are more often recorded than failures (ehrlich, ; veltman et al., ) . the amount of effort directed toward introduction is an important variable affecting successful colonization by vertebrates, and establishment rates increase with high levels of management and numbers of individuals released (ehrlick, ; griffith et al., ; williamson & fitter, b; veltman et al., ) . in contrast, organisms that are casually introduced into new areas have much lower probabilities of establishing and proliferating. this phenomenon is expressed in the tens rule, a statistical characterization of probability outcomes for different levels of invasion success (williamson & fitter, a , b . for a variety of plants and animals, a general rule holds that in species imported (i.e., brought into new areas intentionally or accidentally) appear in the wild, in of those now found in the wild become established, and that in of those established with self-sustaining populations become pests (williamson & fitter, b) . following establishment, proliferation, and rise to pest status, control of exotic vertebrates is often prompted by economic, environmental, or conservation concerns. several control strategies may be pursued, the most common being chemical control (e.g., poisoning) and cultural control (e.g., trapping, fencing, and shooting). the least used option has been biological control. chemical and cultural control of vertebrate pests has been covered by hone ( ) and williams and moore ( ) . biological control is the intentional use of populations of upper trophic level organisms (e.g., predators, parasites, and pathogens) commonly referred to as natural enemies to suppress populations of pests to lower densities than would occur in the absence of natural enemies (debach, ; van driesche & bellows, ) . biological control programs for vertebrates have employed all three classes of natural enemies: predators, parasites, and pathogens. in contrast to weeds and pestiferous arthropods, however, biological control as a population suppression tactic for management of vertebrate pests has historically received much less attention. predators of vertebrates in the few instances they have been used have not been particularly successful. some vertebrate predator introductions also had severe impacts on nontarget organisms. consequently, early unpromising results discouraged intensive development of this technology (howard, ; davis et al., ; wood, ) . there is a need for increased effort using biological control agents against vertebrates, especially where resistance to toxins has developed, or behavior and terrain makes chemical and cultural control difficult and expensive (wood, ; bloomer & bester, ) . biological control should be fostered internationally because many countries experience similar problems (e.g., rabbits are agricultural pests in argentina, australia, chile, europe, and new zealand; rats, cats, and dogs attack endangered faunas on many oceanic islands; feral pigs and goats in new zealand, australia, and the united states degrade habitat and threaten endangered flora). biological control can be aided by the establishment of institutions to help coordinate regional and international research activities. for example, in , the australian federal government supported the creation of the cooperative research center for biological control of vertebrate pest populations (also known as the vertebrate biocontrol center), an unincorporated collaborative venture between state and federal organizations with international cooperators (anonymous, a) . the principle research goal of this institution is population suppression of noxious vertebrate species by regulating reproductive rates (tyndale-biscoe, a). a fundamental issue that has important implications for biological control is understanding the regulatory effects predators have on prey populations. determining impact of introduced predators on pest and nontarget populations is becoming increasingly more important as public awareness of potential nontarget impacts increases and the impact of past introductions on nontarget organisms has become clearer (howarth, ; van driesche & hoddle, ) . introductions of vertebrate predators as biological control agents against vertebrates have in some instances had disastrous impacts on nontarget wildlife, especially insular communities that have lacked an evolutionary history with generalist predators (case, ) . an example is the impact the small indian mongoose on native rail populations in hawaii following its release in the s for rat control (loope et al., ) . the mongoose has little demonstrable effect on rats (cagne, ) and mongoose populations are now poisoned to protect native birds (loope et al., ) . exotic predators may enhance the success of introduced pest species by moderating the competitive impact of natives on introduced pests should predators reduce densities of native species (case, ) . on the other hand, there is very good evidence that under certain circumstances introduced exotic predators can regulate target vertebrate pests. in such cases, predator efficacy may be affected by ecosystem complexity; by influence of such extrinsic factors as weather, disease, or human intervention on pest population growth; and by the availability of alternative food sources to sustain predators when pest populations are low. in simple ecosystems such as islands, establishment of reproducing predator populations can result in the extinction of target pests. in the absence of alternative prey, releases of cats for rabbit control on berlinger island near portugal by a lighthouse keeper resulted in the eradication of the rabbits, and subsequently cats died from starvation (elton, ) . in complex communities, alternate prey may be taken when primary prey populations are low for prolonged periods, as is the case for some microtine rodent species in europe. european rodent populations exhibit fluctuations in size as a response to variation in food availability. these fluctuations are also influenced by predation. population cycles are not observed in southern hemisphere countries where european pests and predators have been transferred (korpim~iki & krebs, ; sinclair, ) . rabbit populations in australia and new zealand are maintained at low levels by introduced predators, but regulation only occurs after pest numbers have been reduced by other means. poisoning programs in new zealand in the s and s substantially reduced rabbit densities and populations were maintained at low levels by introduced predators, in particular, ferrets and cats (newsome, ) . similarly in australia, european foxes and cats maintain rabbit populations at low densities following population crashes caused by prolonged hot summers that reduce forage and browse (newsome et al., ; newsome, ) . mouse populations are regulated in a similar fashion by predators (raptors and foxes) in australia (sinclair et al., ) . introduction of two foxes dusicyon culpaeus (molina) and d. griseus (gray) native to mainland chile onto the chilean side of tierra del fuego island regulated rabbit populations after rabbit densities were substantially reduced by the myxoma virus (jaksic & yhfiez, ) . the suppressive action of predators on rabbits in australia has been demonstrated through predator removal experiments [referred to as perturbation experiments by sinclair, ( ) ] in which european foxes and cats were shot from four-wheel drive vehicles at night. removal of predators resulted in rapid rabbit population growth compared with rabbit densities in control plots in which predators were not removed (newsome et al., ) . the "predator pit" first conceptualized by may ( ) describes rabbit regulation in australia and new zealand by generalist predators. the model suggests that once prey populations fall below certain densities (i.e., because of culling or disease) predators can prevent recovery to higher levels. generalist predators achieve this by maintaining relatively high numbers by attacking alternate prey species, and low but persistent levels of predation on species in the pit prevents pest populations from outbreaking. for the rabbit-fox system in australia, a predator pit operates at densities of to rabbits per kilometer of linear transect. below these densities foxes utilize alternate food sources (e.g., native animals) and above this critical density rabbit populations escape regulation by predators (newsome, ) . in new zealand, rabbits are contained in the pit by cats and ferrets when densities are . rabbits per hectare (barlow & wratten, ) . the effect of predators on long-term population dynamics of alternate prey species is generally difficult to disentangle from confounding effects of habitat degradation and competition for food and breeding sites from other introduced species (pech et al., ) . foxes regulate mice and rabbits through positive density dependence at low prey densities. increasing pest densities during outbreaks results in inverse density-dependent predation and type iii functional responses (sinclair et al., ; pech et al., ) . rabbit and mouse populations escape predator regulation when favorable weather provides good breeding conditions, or when predators are controlled by shooting or poisoning (newsome, ; sinclair et al., ; pech et al., ) . predator control may be necessary for lifestock protection or for conservation of endangered wildlife and when implemented resurgence of pest populations occurs (newsome et al., ; newsome, ) . predator numbers may increase when primary prey (e.g., rabbits) are abundant. predation by abundant predators on secondary prey (e.g., native animals) result, leading to declines in secondary prey density. under such circumstances native prey species may only persist in refugia or in areas with artificially reduced predator pressure (pech et al., ) . altematively, declining densities of a primary pest prey species (either through management or disease) can intensify predator attacks on nontarget organisms. therefore, when conservation of endangered natives is a concern, culling of predators may be undertaken either concurrently with the decline in prey density or in anticipation of such a decline (grant norbury, personal communication, ) . in such situations an integrated approach to managing vertebrate pests and their predators is necessary (newsome, ) . predator efficacy can be enhanced either through habitat modification or resource provisioning. cats can maintain rat and mouse populations around farm buildings below environmental carrying capacity as long as they are provisioned with additional food (e.g., milk). dietary supplementation prevents rodent extermination and the subsequent extinction of cats. sustaining a cat population prevents uncontrolled invasions by rodents and low pest densities are maintained (elton, ) . provision of nesting boxes for barn owls (tyto alba javanica) reduces crop damage by rats in malaysian oil palm plantations; rodenticide use declined, and in some instances use was eliminated (wahid et al., ) . changes in management practices can improve predator efficacy. rodent control by tyto alba (scopoli) in pinus radiata don plantations in chile was enhanced by clearing -m wide strips between trees (for owls to maneuver in while in flight) and construction of perches in forests (for resting and surveillance). bam owl numbers and predation rates on rodents increased following habitat modification (mufioz & murfa, ) . under increased predation pressure, rodents will modify foraging behaviors by reducing activity when owls are flying or making hunger calls (abramsky et al., ) . learning in this manner produces behavioral adaptations because of strong selection pressures to minimize predation risks on pest populations (davis et al., ) . parasites or macroparasites (e.g., helminths, lice, ticks, fleas, and other metazoans) do not typically kill their hosts as a prerequisite for successful development as insect parasitoids do. they tend to be enzootic (i.e., remain at fairly constant levels through time) and usually must pass through a free-living stage to complete an entire life cycle (anderson, ; mccallum, ) . the potential of parasites to regulate vertebrate host populations was first proposed as early as (lack, ) and later was demonstrated theoretically with lotka-voltera models in which parasites increased host mortality rates may, ) . parasites act in a positive density-dependent manner by adversely affecting host survival or reproduction dobson & hudson, ; scott & dobson, ) . host parasite load also affects the ability of individual parasites to grow, reproduce, and survive in definitive hosts; and the severity of density dependence on host and parasite fitness is affected by patterns of parasite distribution in host populations (scott & lewis, ) . helminths, for example, tend to be aggregated within host populations so that few hosts are heavily burdened while most are lightly infected (scott & lewis, ) . density-dependent constraints on parasite survival and reproduction occur in the few heavily infected hosts, and under such conditions, helminth population stability is enhanced . furthermore, parasites with low-to-moderate pathogenicity exert stronger regulatory actions on populations than highly pathogenic species that cause their own extinction by killing hosts before transmission (anderson, ) . parasite regulation of vertebrate populations has been observed under field conditions. botfly (cuterebra grisea coquillett) parasitism of voles [microtus townsendii (bachman)] in vancouver canada, is inversely density dependent; and botfly infestation significantly reduces vole survival, reproduction, and development (boonstra et al., ) . the parasitic helminth trichostrongylus tenuis (cobbold) is the primary agent responsible for long-term population cycles in red grouse [lagopus lagopus scoticus (latham)] inhabiting scottish heathlands (dobson & hudson, ) . the regulatory effect of t. tenuis has been demonstrated by reducing parasite infestations with helminthicides in experimental birds. treated grouse showed increased overwintering survival, clutch sizes, and hatching rates when compared with untreated birds (dobson & hudson, ) . in the laboratory, introduction of the nematode heligmosomoides polygyrus dujardin reduced mice densities by % in comparison with control populations. reduction of nematode transmission rates and elimination of parasites with helminthicides allowed infested mouse populations to increase (scott, ) . although host and parasite densities in this study were higher than those found in nature, the data showed that introduction of a parasite regulated host population abundance. the potential effectiveness of nematodes as biological control agents in field situations has been evaluated for control of the house mouse, an introduced pest in australia (singleton & mc-callum, ; spratt, ) . mouse (mus domesticus) populations erupt every to years in cereal-growing regions of southeastern australia (singleton & mccallum, ; mccallum, ) and economic losses to mouse plagues exceed $ million (australian) (beckman, ; singleton, ) . outbreaks are associated with high autumn rainfalls following prolonged periods of drought that extend the growing season for grasses that set seeds. this high-quality food source increases high mouse survivorship and breeding throughout winter. population crashes occur when food supplies are exhausted (singleton, ) . saunders and giles ( ) suggested that droughts are necessary to remove the regulating effects of natural enemies, and this removal combined with favorable weather conditions permitted mouse numbers to increase rapidly. capillaria hepatica (bancroft), a parasitic nematode that infests mice, is naturally occurring and widely distributed in pestiferous rodents in coastal areas of australia. it is, however, absent in mouse populations in cereal-growing areas . this nematode is unique because it is the only known helminth with a direct life cycle that requires host death for transmission. female nematodes deposit eggs in the host's liver; and these eggs are liberated by predation, cannibalism, or necrophagy with subsequent digestion of infected liver. unembryonated nematode eggs voided after ingestion undergo embryonation to become infective and are probably consumed when mice preen their fur and feet (fig. ) . ground beetles (carabidae) may vector c. hepatica eggs after they have been eaten (mobedi & arfaa, ). firststage larvae emerge from ingested embryonated eggs and move into the liver through the hepatic portal system (wright, ) . nematode infestation significantly reduces natality and numbers of young mice weaned by infected females (mccallum & singleton, ; singleton & mc-callum, ; . capillaria hepatica is associated with introduced rat and mouse species in urban areas, and naturally occurring infections in native australian mammals are rare probably because of the susceptibility of nematode eggs to ultraviolet radiation and desiccation singleton et al., ) . native australian mice and marsupials are susceptible to experimental infection in laboratories . rats (r. norvegicus and r. rattus) are major reservoirs for c. hepatica in urban areas and infestation rates range from to % (childs et al., ; singleton et al., ) . infestation levels are lower in sympatric mice populations ( to %, singleton et al., ) . low rat numbers in cereal-growing regions of australia may be a factor contributing to the nonpersistence of c. hepatica in these areas . infestation of nonrodent mammals by c. hepatica is rare but has occurred in rabbits (gevrey & chirol, ) , dogs (leblanc & fagin, ) , horses (munroe, ) , and humans (pannenbecker et al., ) . human infections can be treated successfully (pereira & franca, ) . exploratory models investigating the impact of c. hepatica on mouse populations indicated that the requirement of host death for parasite transmission is strongly destabilizing. in the absence of resource limitation mouse densities increase similarly to disease-free populations before parasites have an impact and infected populations decline in density (mccallum & singleton, ) . slow regulation of mouse populations occurs because of the need for host death for transmission. consequently, the nematode's life cycle operates on the same time scale as that of its host instead of being orders of magnitude faster, as is the case with other parasites that do not require host death for transmission (mccallum & singleton, ; singleton & mc-callum, ) . the destabilizing influence of c. hepatica on mouse parasitology today, , [ ] [ ] [ ] [ ] populations may contribute to localized host and parasite extinctions. these extinctions, coupled with very low mouse densities in nonoutbreak years, result in population bottlenecks and may explain why nematodes do not persist in regions where mouse outbreaks occur. soil type, temperature, and moisture content do not affect nematode egg survival and embryonation under favorable conditions in outbreak regions (spratt & singleton, ) . outbreak intensity can theoretically be reduced by c. hepatica if populations are inoculated early, preferably year before an outbreak is expected (mccallum, ) . releases of high doses of nematode eggs in the summer or autumn when mouse densities are sufficient to enable high levels of transmission may offer the best chance for successful control (mccallum & singleton, ; mccallum, ) . field experiments in enclosures and with increasing populations of free-ranging mice have failed to demonstrate long-term regulation on mouse population growth with periodic inoculative releases of c. hepatica eggs. unexpected declines in control populations (i.e., populations not treated with nematodes) have to some degree masked the effect of c. hepatica on mice populations singleton et al., ; singleton & chambers, ) . transmission of c. hepatica in treated populations is not density dependent and can occur at low levels for to months. transmission rates show seasonal trends influenced by soil temperatures and increasing aridity singleton & chambers, ) . improved understanding of the influence of factors (such as temperature and rainfall on nematode persistence, survival, and transmission in field situations) and timing of releases of parasite eggs may improve releases of c. hepatica for control of mouse outbreaks (singleton et al., ; singleton & chambers, ) . vertebrate species that successfully colonize new habitats have reduced parasite loads in comparison with mother populations from which they originated (dobson & may, ) . lower infestation levels probably occur because individuals that make up small founding populations either were uninfected or had only a limited subset of the total potential parasite species found in the area of origin, or intermediate hosts required for parasite persistence were absent in the new range. sparrows and starlings, both successful colonizing species from europe, have two to three times fewer parasites in north america compared with populations from which they originated. populations established outside of europe may have benefited from reduced parasite burdens, although there are no quantitative data to indicate that this aided establishment and proliferation (dobson & may, ) . investigating the role of parasites on population dynamics of rabbits in europe with the view for possible introduction into countries where rabbits are pests is also warranted (boag, ) . introduced mammals such as rats, goats, and cats on oceanic islands exhibit depauperate parasite faunas (dobson, ) . fewer parasites coupled with presumed low genetic diversity of small founding populations, and reduced selection pressures for parasite resistance may make these pest vertebrates vulnerable to introduced host-specific parasites. the ideal parasite introduced into a high-density pest population that originated from a small founding population should have low-to-intermediate pathogenicity, because such parasites establish and maintain themselves in populations at lower densities than more pathogenic species do . macroparasites that reduce both host longevity and fecundity may have the potential to cause sustained reductions of host population densities (dobson, ) . low genetic variability among target populations should theoretically enable introduced parasites to become more evenly distributed among hosts, and reduction in parasite aggregation would increase natural-enemy efficacy (dobson & hudson, ) . the possibility of reassociating parasites with vertebrate pests is not limited to mammals and birds. host-specific parasites may have the potential to reduce reproduction and longevity of pest reptile (dobson, ) and amphibian species (freeland, ) . the brown tree snake is the proximate cause of native bird extinctions on guam following its accidental introduction after world war ii on military equipment (pimm, ; savidge, ; jaffe, ; rodda et al., ) . the snake also has caused declines of native reptile and small mammal populations, and enters houses and attacks sleeping human infants (rodda et al., ) . additionally, the brown tree snake has caused eco-nomic losses by adversely affecting domestic animals (e.g., chickens and pets), and high densities of snakes on power lines regularly cause short circuits that interrupt electrical supplies and necessitate repairs. control of the brown tree snake has been attempted through trapping, but the snake's extreme preference for live bait over artificial lures has made this approach impractical (rodda et al., ) . the brown tree snake mnative to eastern indonesia, the solomon islands, new guinea, and northeastern australiambelongs to the family colubridae. it is the only member of this family on guam. there is one native species of snake on guam, the blind snake, rhamphotyphlopys braminus (daudin), which belongs to the family typhlopidae and is the only snake occurring on many islands in the central pacific region (t. fritts, personal communication, ) . the brown tree snake has extended its range and is now established on the previously snake-free island of saipan, and this snake has been intercepted in hawaii; corpus christi, texas, and spain (rodda et al., ) . given the propensity for the brown tree snake to be dispersed to new habitats within cargo loads on planes and ships, the major social, economic, and ecological problems that are caused on islands after colonization, in addition to its distant taxonomic relationship to snakes common to pacific islands, make the brown tree snake an excellent target for biological control. the taxonomic relationship between colubrids and typhlopids may simplify the task and reduce the cost of finding natural enemies unique to the brown tree snake. parasites or pathogens that are host specific just to the family (i.e., colubridae) or genus (i.e., boiga) level may be safe to nontarget snakes (e.g., typhlopids) because these organisms have not evolved the ability to cause disease in distantly related hosts. extreme caution should be exercised when implementing a biological control program against vertebrate pests with parasites. parasites and pathogens can pose major threats to populations of endangered animals (mccallum, ; mccallum & dobson, ) . the susceptibility of nontarget organisms, especially endemic species, to infection by candidate biological control agents should be investigated thoroughly prior to parasite releases. reassociating parasites that preferentially infect a competitively dominant pest species may increase species diversity of invaded communities by reducing the pest's prevalence. in this instance, the natural enemy would assume the position of a keystone parasite (marcogliese & cone, ) . pathogens or microparasites include viruses, bacteria, and protozoans. pathogens tend to be unicellular and exhibit epizootic (i.e., boom or bust) life cycles due to rapid proliferation in hosts (anderson, ; mccallum, ) . the potential of pathogens to regulate vertebrate population densities by reducing the longevity and fecundity of infected hosts has been demonstrated theoretically with mathematical models and by perturbation experiments using vaccines (smith, ) . as with macroparasites, models indicate that microparasites of intermediate pathogenicity are more effective biological control agents (anderson, ) . highly virulent pathogens kill themselves by destroying hosts before they can be transmitted and avirulent strains are not transmitted because they are removed by the immune system. the immune system is theorized as being responsible for maintaining the intermediate virulence of vertebrate microparasites (anita et al., ) . pathogens that are readily transmitted (i.e., microparasites spread by water, air, and vectors) or have high-density host populations are more contagious than those with low transmission rates (i.e., spread is by host-to-host contact) or low host densities (ebert & herre, ) . new associations between pathogens and novel hosts are generally not more harmful than those that have evolved closely with the host. experimental evidence indicates that novel disease-causing organisms are on average less harmful, less infectious, and less fit than the same parasite strain infecting the host it is adapted to (ebert & herre, ) . also, a microparasite's ability to infect and exploit novel hosts decreases with increasing geographic and presumably genetic distance from the host to which the pathogen is adapted (ebert, ) . exceptions do occur, however, and pathogens can have devastating impacts on hosts that have no evolutionary history with the disease organism. an example is the myxoma virus, the causative agent of myxomatosis in european rabbits. the use of this natural enemy against rabbits in australia and europe has been the most thorough biological control program against a vertebrate pest. the myxoma virus is a member of the genus leporipoxvirus (poxviridae) and originated from south america where it was first recognized as an emerging disease of european rabbits in laboratories in montevideo, uruguay, in . infected laboratory rabbits died of a fatal febrile disease that caused tumors on the head and ears. the tumors resembled myxomas (a benign tumor composed of connective tissue and mucous elements) and the disease was subsequently named infectious myxomatosis of rabbits (fenner & marshall, ; fenner & ratcliffe, ; fenner, ) . the indigenous host for myxoma virus in south america is the forest rabbit [sylvilagus brasiliensis (linnaeus)]. unlike its effect on european rabbits, myxoma inoculum injected into forest rabbits caused benign fibromas at the site of inoculation that persisted for many months, although death did not occur. mosquitoes were implicated in vectoring the disease from forest rabbits to european rabbits being bred in south american rabbitries. another leporipox-virus has been isolated in california from the brush rabbit, sylvilagus bachmani (waterhouse), and is closely related to the myxoma virus (fenner & marshall, ; fenner & ratcliffe, ; fenner, ; fenner & ross, ; ross & tittensor, ) . myxoma virus has been used in australia, europe, chile, and argentina for biological control of european rabbits. the virus was first imported into australia from brazil in and but was not released (fenner & ratcliffe, ) . work by australians with the virus began again in the united kingdom in and continued with caged rabbits on wardang island off the south coast of australia. the virus was successfully established on mainland australia in (fenner, ) and within years it had established itself over most of the rabbit's range (fen-ner& ratcliffe, ) . the virus initially had a major impact on the estimated million rabbits and on the damage they caused, reducing population density by to %. efficacy was dependent on climate and rabbit population susceptibility. populations have subsequently increased and stabilized at around million because of myxomatosis. damage attributable to rabbits still amounts to $ million (australian) annually, including both lost agricultural production and cost of control applications (robinson et al., ) . in addition to agricultural losses, rabbits severely affect native flora by eating foliage and inducing wind and water erosion of soils by overgrazing. native fauna are also affected as rabbits out-compete indigenous herbivores and dense rabbit populations sustain exotic predator populations that feed on native animals (gibb & williams, ; myers et al., ; robinson et al., ) . within a few years of the initial panzootic, field isolates of the virus showed less virulence when compared with the original strain that had been released. the original strain killed > % of laboratory rabbits on average . days after infection, while circulating strains caused % mortality after . days. genetic resistance in rabbits was also detected (fenner & marshall, ; fenner & ratcliffe, ) . dual natural selection had occurred, the virus had attenuated, and rabbits had increased in resistance to the disease. mosquitoes have been responsible for vectoring myxoma virus in australia. the european rabbit flea, spilopsylus cuniculi (dale), an important vector in europe, was introduced into australia in and increased the geographic distribution of the disease. this flea did not persist in areas with rainfall < mm. the xeric adapted spanish rabbit flea, xenopsylla cunicularis smit, was introduced in and active redistribution is still ongoing (fenner & ross, ) . new zealand also has inordinate numbers of rabbits, and attempts to establish the myxoma virus from to failed because of inclement weather and a paucity of suit-able arthropod vectors. further attempts at establishment were not undertaken because poisoning programs had reduced rabbits to very low numbers, additional control expenditure was unjustifiable, and the new zealand public was not in favor of using lethal myxoma virus for rabbit control on humanitarian grounds (gibb & williams, ) . until the s, myxomatosis was the only disease known to severely affect rabbit numbers. a second highly contagious viral disease emerged in the mid- s and was accidentally introduced onto mainland australia (o'brien, ) . it is the first pathogenic natural enemy to have established in new zealand for biological control of rabbits. rabbit calicivirus disease (rcd) [also known as rabbit hemorrhagic disease virus (rhdv)] emerged as a fatal disease in in angora rabbits exported from east germany to jiangsu province of china (liu et al., ) . in , the disease appeared in italy where million rabbits were estimated to have died. the disease spread rapidly through rabbit populations in europe reaching the united kingdom in (chasey, ) . the probable mechanism for dispersal in continental europe was the movement of live rabbits and rabbit products. transmission of rcd from france into coastal areas of southeast england is thought to have occurred by wind-borne aerosols containing virus, birds, and transchannel ferry traffic (chasey, ) . outbreaks of rcd occurred in mexico in and (gregg et al., ) and in rrunion island in the indian ocean in . movement of rcd to these areas probably occurred with imports of frozen rabbit carcasses from china because the virus can survive freezing to temperatures of - ~ (chasey, ) . the rcd virus belongs to the caliciviridae and consists of a positive sense, single-stranded rna genome, enclosed by a sculptured capsid composed of multiple copies of a single major protein of kda, and is to nm in diameter (ohlinger et al., ; parra & prieto, ) . disease symptoms are characterized by high morbidity and mortality in rabbits over weeks of age. younger rabbits often survive infection and may develop antibodies to rcd virus (nagesha et al., ) . clinically, rcd symptoms are expressed after an incubation period of to h in which a febrile response and increasing lethargy are observed. infected rabbits typically die within to h postinfection and % mortality is observed after days. necropsies show a pale swollen friable liver, enlarged spleen, and clots in blood vessels. death is ascribed to acute necrotizing hepatitis and possible hemorrhaging (fuchs & weissenbrck, ; studdert, ) . however, necropsies close to the time of death show an absence of hemorrhaging and inclusion of hemorrhagic in the name of this rabbit disease that indicates the cause of death is misleading (studdert, ) . a different viral disease is responsible for european brown hare syndrome (ebhs) which causes severe hepatic necrosis in hares (lepus europaeus pallas and l. timidus linnaeus). the disease was first recorded in sweden in , and spread through continental europe and reached the united kingdom in (fuller et al., ) . in sweden, losses of hares to ebhs occurred years prior to sympatric rabbit populations developing rcd. similar observations were made in the united kingdom where hares began dying from ebhs years before rcd was observed in rabbit populations (fuller et al., ) . electron microscopy, nucleotide sequencing, and experimental cross-transmission studies have indicated that rcd virus and ebhs virus are closely related (le gall et al., ) but distinct members of the caliciviridae (chasey et al., ; nowotny et al., ) . disease symptoms are generally similar for rabbits and hares but show distinguishing characteristics in necrosis of liver lobules and clotting of blood vessels (fuchs & weissenbrck, ) . serological studies on rabbit sera collected in from czechoslovakia and austria indicate that rcd virus probably evolved from an apathogenic strain endemic to europe from at least this time (nowotny et al., ) . studdert ( ) speculates that the causative agent of rcd probably existed in europe as a quasi species, a collection of indifferent mutants with a variety of accumulated nucleotide changes. in this scenario, mutants occupied a specific ecological niche until one strain better adapted to prevailing conditions became the dominant member of the population. adaptation may have occurred because mutations caused increased virulence in an avirulent rabbit virus or increased the host range of hare-infecting viruses by allowing mutant strains to bind more efficiently to surface receptors on rabbit hepatocytes. given studdert's ( ) speculative scenario, rcd virus may be a highly evolvable organism. european rabbits appear to be the only animals susceptible to infection by rcd virus, and vaccines have been developed to protect domestic animals (boga et al., ) . other rabbits including cottontail rabbits (sylvilagus spp.), black-tailed jack rabbits (lepus californicus gray), volcano rabbits [romerolagus diazi (ferrari-prrez)] (gregg et al., ) , and hares (gould et al., ) are not affected. the limited host range of rcd virus makes it an obvious candidate for use in a biological control program against european rabbits in new zealand and australia. a joint biological control program between these two countries using rcd virus was initiated in and a strain of virus from the czechoslovakia republic was imported into australian quarantine facilities in to test effects on nontarget species (robinson & westbury, ) . host-specificity testing of nontarget species in australia for susceptibility to rcd virus further verified the limited host range of this natural enemy. test subjects in-cluded domestic lifestock (horses, cattle, sheep, deer, goats, pigs, cats, dogs, and fowls) , noxious exotic vertebrates (foxes, hares, ferrets, rats, and mice), native mammals (eight species), birds (five species), and reptiles (one species). there was no evidence for viral replication, clinical signs, or lessions in any organisms tested (gould et al., ) . artificial inoculation of rcd virus in north island brown kiwis (apteryx australis mantelli bartlett) and lesser short-tailed bats (mystacina tuberculata gray), both native to new zealand, also failed to produce disease symptoms (buddle et al. ) . the apparent host specificity of rcd virus to rabbits, rapidity of action, and the capacity to infect rabbits from other rabbits, [through feed and feces, or from a contaminated environment (o'brien, ) ] prompted further evaluation of this biological control agent under field quarantine conditions in australia. studies monitoring the effects of rcd virus on rabbit populations were initiated on wardang island near adelaide off the south coast of australia in (rudzki, ; robinson & westbury, ) . in september , rcd breached quarantine and appeared on mainland australia, possibly carried there by calliphorid flies and onshore winds (cooke, ; lawson, ) . attempts at containment failed (seife, ) . within months of the initial discovery of rcd virus on the mainland, an estimated million rabbits were killed in south australia. in dry areas, to % of infected populations died (anderson, ) with dead rabbits averaging per hectare. elsewhere, fatality rates were closer to % (anonymous, b) . in the period from october to november , an estimated total of million rabbits died from rcd in south australia and the majority of surviving rabbits were less than weeks of age (cooke, ) . the development of resistance in young rabbits may have profound effects on the long-term population dynamics on the rabbit-rcd virus system. ten arthropod vectors of rcd virus have been identified and include flies, mosquitoes, and rabbit fleas (anonymous, b) . rates of spread of rcd are greatest in spring and autumn at to km a day and are correlated with peaks of insect activity. dispersal of the disease probably has been assisted by humans moving contaminated material to new areas (cooke, ) . increased attacks on native fauna by exotic predators such as foxes because of declines in rabbit numbers do not appear to have occurred because predator populations have declined with rabbit numbers (anonymous, b). the virus is now endemic in australia and will probably be officially declared as a biological control agent under the biological control acts of the commonwealth and states (robinson & westbury, ) . rcd virus was smuggled into the south island of new zealand by high country farmers in august and illegally disseminated by feeding rabbits carrots and oats satu-rated with contaminated liquefied rabbit livers. a network of cooperators spread the virus over large areas of the south island and its subsequent spread (human assisted through the movement of carcasses, baiting, and insect vectors) made containment and eradication of the disease impossible. such actions by farmers clearly violated new zealand's biosecurity act, which was enacted in part to protect agriculture from unwanted introductions of pests. the new zealand government has sanctioned controlled virus releases into new areas. the short-term impact of rcd on new zealand rabbit populations has resulted in to % mortality in central otago and large-scale field studies are planned (g. norbury, personal communication, ) . cats on oceanic islands have been subjected to biological control with pathogens. six cats were introduced onto marion island in the indian ocean in (howell, ) ; by , numbers were in excess of and were increasing an average of % per year (van rensburg et al., ) . populations were sustained by consuming approximately , seabirds yearly and cats were probably responsible for the local extinction of the common diving petrel pelecanoides urinatrix (gmelin) (bloomer & bester, ) . surveys of cats on marion island revealed the presence of feline herpes virus and feline corona virus, but the highly contagious feline parvo virus was absent in the population (howell, ) . initiation of a biological control program with feline parvo virus, the causative agent of feline panleucopenia, began in with the release of artificially inoculated feral cats collected from the island (howell, ) . the disease reduced cat numbers by % after years by reducing fecundity and increasing mortality of juvenile cats (van rensburg et al., ) . virions found in high concentrations in feces, urine, saliva, and vomit were transmitted through direct contact between cats or contact with contaminated objects (howell, ) . annual declines of cat numbers were % from to . this rate decreased to % per year from to and was accompanied by lower titers of virus in serum samples collected from feral cats, indicating that viral efficacy was decreasing (van rensburg et al., ) . at reduced densities, hunting and trapping became viable and have been incorporated into an ongoing eradication program that may be assisted by the use of trained dogs (bloomer & bester, ) . sexually transmitted diseases have adverse effects on domestic and wild vertebrates by reducing survival, conception rates, and numbers of offspring born and successfully weaned (smith & dobson, ) . rabbits are susceptible to infections of venereal spirochetosis (treponema cuniculi), which causes sterility (smith & dobson, ) . goats can develop trichomoniasis, a sexually transmitted disease caused by the flagellated protozoan trichomonas foetus (reidmuller). this pathogen has been suggested as a bio-logical control agent for goat populations on oceanic islands that lack this microparasite (dobson, ) . sexual transmission of diseases may further guarantee host specificity in biological control programs. it also enhances the ability of parasites and pathogens to persist in low-density populations or solitary species (e.g., predators). the rate of spread of sexually transmitted organisms is tightly correlated with mean and variance of the numbers of sexual partners per host because of the need for host-tohost contact (horizontal transmission) for transmission. host population density is not important with respect to persistence or rate of spread of sexually transmitted diseases. this property, coupled with asymptomatic carrier states, long infectious periods, or vertical transmission (infective propagules are passed from mother to offspring), greatly enhances the ability of pathogens to persist in lowdensity host populations (smith & dobson, ) . because sexually transmitted organisms can persist in low-density populations or populations of declining density, the potential of genetically engineering sexually transmitted viruses to sterilize infected hosts is being investigated (barlow, ) . viruses that have antigens from the host sperm, or the zona pellucida around host eggs engineered into the genome provoke an immune system response that renders the recipient sterile. immunocontraception (also referred to as immunosterilization) as a means to control noxious vertebrates is being actively pursued by australia and new zealand (mccallum, ) . an alternative approach to immunocontraception is to use genetically modified microparasites to prevent lactation in females so that juveniles are not successfully weaned or to interfere with hormonal control of reproduction (cowan, ; jolly, ; rodger, ) . many species that become pests are distinguished from nonpestiferous species by their higher intrinsic rates of increase (rm). pest vertebrates have high rm values characterized by large litters, and by maturing sexually at young ages. agents that reduce reproductive rates may be more effective for control than mortality-inducing biological control agents are because resistance development should take longer to occur and population recovery would be slower (tyndale-biscoe, b) . resistance development may be further delayed by combined use of multiple agents that affect fertility in different ways [e.g., using agents that cause sterilization, alter levels of reproductive hormones, or affect lactation (cowan & tyndale-biscoe, ) ]. in sexually reproducing vertebrates, proteins associated with male and female gametes are potentially foreign antigens in the opposite sex. exposure to reproductive antigens occurs when females receive sperm and accessory fluids from males during copulation. as a general rule, females do not develop antibodies to these antigens because physiological and immunological mechanisms have evolved to prevent this (robinson & holland, ) . inoculation of sperm into females of the same species either subcutaneously or intramuscularly produces high sperm antigen antibody titers in recipients. in most cases, this causes either permanent or temporary infertility in females. such results indicate that sperm antigens in the reproductive tract are tolerated and that exposure to these antigens by different routes overcomes protective mechanisms, with infertility resulting (robinson & holland, ) . sperm antibodies in females that can arise from either systemic or local immune responses are found in cervical mucus, genital fluids (e.g., endometrial, tubal, and follicular fluids), and blood. antibodies bind to sperm, often in specific locations such as the head, midpiece, tail shaft, or tail tip. once bound to sperm, antibodies cause agglutination (e.g., irreversible binding to cervical mucus that normally aids sperm transport) or immobilization of sperm. antibodies may also interfere with acrosome reactions preventing ovum penetration and fertilization, or they block the binding of sperm to the zona pellucida (shulman, ) . the zona pellucida (zona) that surrounds growing oocytes and ovulated eggs is antigenic and available to circulating antibodies during oocyte growth and ovulation. nonreproductive tract inoculation of females with zona preparations leads to infertility (millar et al., ) . antibodies produced in response to administered zona antigens bind to the zona and prevent sperm penetration (millar et al., ) . zona glycoproteins are highly conserved among mammals, for example, nonspecific pig zona preparations cause infertility in humans, primates, dogs, rabbits, horses, and deer (robinson & holland, ) . a major objective in immunocontraception research is isolation of species-specific zona glycoproteins that do not cause sterility induced by immune response in species from which zona preparations were not derived. low variability among zona glycoproteins may limit the number of species-specific zona preparations for immunocontraception (millar et al., ) . immunocontraception for wildlife population control has been successfully implemented for horses (equus caballus linnaeus) (kirkpatrick et al., (kirkpatrick et al., , . free-ranging feral mares inoculated by dart gun with porcine zona pellucida showed depressed urinary estrogen concentrations and failure to ovulate. zona booster inoculations given years after initial inoculations prevented conception in treated horses for a third year compared with control populations that were not vaccinated. contraceptive effects were reversible after years of consecutive treatment, but prolonged treatment ( to years) with zona preparations caused irreversible ovarian dysfunction and fertility loss (kirkpatrick et al., (kirkpatrick et al., , . similar results have been achieved with porcine zona pellucida inoculations in whitetail deer [odocoileus virginianus (zimmerman)] (kirkpatrick et al., ) . gametic antigens that induce immune response can be administered by baits that are ingested by target organisms or can be inoculated directly into hosts with darts or bullets (tyndale-biscoe, b). injection of foxes with sperm antigens reduces fertility from to %. baits are considered the favorable method for delivering antigens to foxes in australia. potential baits include dried meats that contain microencapsulated antigens. use of recombinant bacterial vectors (e.g., salmonella typhimurium) also are being considered. an orally administered agent needs to reach the lower gastrointestinal tract to stimulate a response in the common mucosal immune system in the gut-associated lymphoid tissue. this in turn induces mucosal immunity in the reproductive tract of female foxes and causes sterilization (bradley et al., ) . at present, an effective bait specific to foxes that is environmentally stable and easy to manufacture has not been developed. nontarget impact is a concern because most antigens exhibit some specificity to the family level only. effective vaccines for rabies have been delivered as oral baits to foxes in europe, demonstrating the baiting technique is an effective dissemination method (bradley et al., ) . models indicate density-independent factors such as drought and rain (which affect pasture growth and rabbit numbers) strongly influence the effectiveness of baitdelivered fertility control in reducing fox abundance . an alternative proposal to deliver antigens orally is to develop transgenic plants to produce and deliver gametic antigens in palatable form to herbivorous pests. plants could be sown over target areas and allowed to become self-propagating vaccines. transgenic seeds, fruits, or leaves (e.g., transgenic carrots or maize) could be harvested and used as oral baits delivered to specific sites such as fenced watering points that allow pest animals access while excluding lifestock (smith et al., ) . baiting is an expensive form of control that requires monitoring of dosage and uptake rates and multiple areawide applications. problems of hormonal modification of behavior and delayed population control are additional drawbacks. one advantage is that baits can be used to treat localized populations that are problematic. similar shortcomings exist with antigen inoculations by projectiles where cost estimates are significant. to control the estimated , wild horses in australia with dart-delivered porcine zona pellucida would cost $ (australian) per horse per year compared with cents for permanent control with a bullet (tyndale-biscoe, ) . lethal methods of control provide immediate impact on pest populations and reduce pest status rapidly, with control being quickly observable. in contrast, fertility impairment is not immediate, population responses are delayed, and large proportions of populations need to be sterilized for this technique to be effective. large-scale distribution of gametic antigens might be possible through releases of host-specific microparasites expressing species-specific antigen genes (tyndale-biscoe, a , b . self-spreading and replicating parasitic vectors that have been genetically engineered and that may require periodic reinoculation into populations are analogous to augmentative biological control programs with traditional natural enemies (e.g., parasitoids, predators, or pathogens) released periodically for the control of pest arthropods. host-specific viruses carrying foreign dna could be cheap and effective biological control agents that have the potential to disseminate widely by sexual transmission, contagion, or arthropod vectors. the selected micro-or macroparasite must be able to carry foreign dna coding for gametic antigens, as well as promoters to express foreign genes and cytokines to enhance effectiveness (tyndale-biscoe, b). such agents must be able to reduce growth rates of infected populations and to maintain reproductive rates at lower levels (caughley et al., ) , and should not interfere with sexual behavior or social organization (caughley et al., ; robinson & holland, ; tyndale-bisoce, b) . with some pests such as rabbits and foxes, dominant members of populations make the main contribution to reproduction and inhibit breeding by subordinate members by occupying prime territories. ideally, a sterilizing agent should not change social hierarchies by allowing individuals with lower social status to successfully rear more offspring because this will cause pest populations to increase substantially (caughley et al., ) . genetically engineered agents should sterilize females because models predict greater population suppression with infertile females than with sterilized males (barlow, ; caughley et al., ) . in the absence of arthropod vectors, sexually transmitted diseases engineered to cause sterilization are superior to nonsexually transmitted ones because multiple matings with sterilized females increases contact rates and the competitive ability of the engineered agent with nonsterilizing strains. the impact of immunocontraception is further enhanced if the sterilizing agent causes limited host mortality and there is low naturally occurring immunity to sexually transmitted diseases (barlow, ) . sexually transmitted herpes-type viruses are being proposed as vectoring agents to induce sterilization in brushtail possums in new zealand (barlow, ; barlow, ) . the recently identified borna disease virus that causes wobbly possum disease in new zealand may be a suitable alternative to a herpes virus (atkinson, ) . the myxoma virus and murine cytomegalovirus are being investigated as gamete antigen delivery agents for rabbits and mice, respectively, in australia (mccallum, ; tyndale-bisoce, b; shellam, ) . four potential insertion sites for genes coding for gametic antigens have been identified in myxoma virus and recombinants have been constructed to express two esherichia coli (escherich) enzymes and influenza virus hemagglutinin genes. the ability of a novel myxoma virus to compete and spread among existing myxoma strains in field situations has been demonstrated by monitoring the spread of virus containing identifiable gene deletions (robinson et al., ) . the myxoma virus that can express foreign genes may operate as a vector for gametic proteins (robinson et al., ) . work is continuing on isolating and inserting rabbit gamete antigen genes into the myxoma virus genome (robinson et al., ) . the responses of experimental rabbit and fox populations in australia to imposed sterility by surgical ligation of fallopian tubes in females have been studied in an attempt to simulate the effects of virally mediated immunocontraception after recombinant virus establishment in wild populations. this technique prevents conception among predetermined proportions of females in populations without interfering with hormones or reproductive behavior (williams & twigg, ) . the dynamics of rabbit populations enclosed by rabbit-proof fencing that exhibited , , , or % sterilization of females were studied in each of two locations in western and eastern australia where climate patterns differed. females born into treatment populations were trapped and sterilized to maintain the same overall sterility levels (williams & twigg, ) . juvenile rabbits born into populations with sterilized females exhibited greater survivorship because of lowered competition for resources. this greater survival compensated for decreased fertility, but recruitment rates were ultimately constrained by environmental factors (e.g., depletion of vegetation). in populations with % sterility, reduced juvenile mortality did not compensate fully for lowered reproduction, smaller numbers of rabbits were recruited into these populations, and numbers subsequently declined. these results indicate that levels of sterilization with a genetically altered micorparasite have to reach at least % to achieve reductions in population density (williams & twigg, ) . surgical sterilization does not affect reproductive behavior in treated populations. sterile dominant female rabbits maintain hierarchical dominance, increased body weight over control females, continued to defend prime territory, and engaged in normal reproductive behavior including breeding burrow construction (tyndale-bisoce, b). birth rates of sexually mature females were in direct proportion to the level of fertility in experimental populations, indicating that fertile females did not respond to female infertility or decreased densities of young by producing larger litters (williams & twigg, ) . sterilized females tended to live longer than unsterilized females. this increased longevity suggests that sterile females may proportionately increase as treated populations reach an equilibrium density. obviously, larger proportions of sterile females reduce population productivity and the numbers of fertile females that a sterilizing microparasite would have to infect and sterilize. higher proportions of sterile females may reduce numbers of infective individuals harboring sterilizing microparasites and numbers of vectors (e.g., fleas that would spread an engineered myxoma virus), and may contribute to decline of transmission rates. these interactions need to be clarified and mathematical models may be of use here (williams & twigg, ) . engineered microparasites that sterilize pest animals offer the possibility of humane control without killing or causing animals to suffer the effects of debilitating disease. as a form of biological control, immunocontraception may also reduce the need for broadcast distribution of toxins for pest suppression, thereby reducing environmental contamination and nontarget mortality. this is of special concern when pests inhabit suburbs, urban parks, government and state campuses, nature reserves, military bases, or other areas where lethal controls may no longer be legal or safe (kirkpatrick et al., ; williams, ) . the concept of virally mediated immunocontraception has generated considerable debate on legal and ethical issues concerning releases of engineered microorgansims into the environment. once contagious recombinant agents that cause permanent sterilization in animals are released into the environment they cannot be recalled (tyndale-bisoce, ) . several potential risks are recognized. first, engineered viruses that are host specific and contain species-specific antigens could mutate and infect and sterilize nontarget species after release (anderson, ) . under such conditions it may be difficult if not impossible to contain and eradicate a mutant virus from an infected animal population that is abundant, secretive, and free ranging. second, sterilizing viruses either might cross international boundaries accidentally or be maliciously moved to sterilize desirable organisms in new areas (tyndale-bisoce, b) . for example, viruses engineered with little host specificity to sterilize widely dispersed marsupial pests in new zealand may enter australia and infect endangered wildlife (rodger, ; mccallum, ) ; engineered myxoma viruses may spread from australia into the americas and sterilize native rabbit species (tyndale-biscoe, ) . third, dart-delivered contraceptives used for wildlife control in the past have had adverse effects on individuals within target populations. changes in morphology of repro-ductive organs, secondary sexual characteristics, and behavior have been observed. viruses that induce sterility could alter genetic profiles of target populations because infectious agents may act as a new reproductive disease and individuals may exhibit differential susceptibility (nettles, ) . fourth, public concerns over the use of viruses and genetic engineering indicate substantial apprehension about the use of sterilizing viruses for pest management, these fears that need to be fully alleviated may delay or prevent field trials and widespread application (lovett, ) . despite potential drawbacks, immunocontraception is a potentially cost-effective method for reducing pest impact on endangered native species (sinclair, ) and on agricultural yields, and is an additional tool for sustainable pest management (williams, ) . a sterilizing agent that does not cause painful disease symptoms is an ethically acceptable form of pest control that is justifiable from animal rights perspectives, because it does not cause the suffering typical of current lethal methods (e.g., trapping, shooting, poisoning, and introduced disease) ( oogjes, ; singer, ) . under certain circumstances, the use of vectors to disseminate genetically engineered viruses is warranted (mccallum, ) . experience with the myxoma virus in australia indicates that it has not been deliberately or accidentally spread to any other country since its introduction in the s because of either the lack of suitable arthropod vectors or the inability of the virus to establish where different strains are already present. this history may indicate possible difficulty for unintentional establishment of genetically engineered microparasites in new areas, and establishment of engineered myxoma viruses may be possible only with carefully timed and repeated releases into rabbit populations (tyndale-biscoe, ) . however, such safeguards may be moot if a highly competitive sterilizing strain is engineered and released. quarantine legislation designed to prevent accidental or intentional but illegal importation of unwanted organisms would be exercised by countries under current international obligations and should impede establishment in new countries if rigorously enforced. however, current legal safeguards may be insufficient. new zealand's experience with rcd indicates it is possible for lay people to illegally import and establish reproducing populations of exotic pathogens. in australia, rcd breached a carefully planned quarantine on an offshore island. unintended establishment and proliferation of engineered viruses may be contained if outbreaks are recognized early, and if proportions of susceptible individuals are removed rapidly from the population either by culling or by immunizing against the pathogen (tyndale-b iscoe, ) . this has never been tried with wild animal populations. the containment of contagious pathogens, such as foot and mouth disease in livestock, indicates such an approach may be possible. highly attenuated forms of myxoma virus are used to protect wild and domestic rabbits in france and the united states, indicating the availability of such technology for this virus at least (fenner & ross, ; tyndale-biscoe, ) . limited field trails with sterilizing microorgansisms are unlikely before (anderson, ). there is abundant evidence that introduced exotic vertebrates that establish feral reproducing populations have disastrous consequences for agriculture and preservation programs for native plants and animals. sources of current vertebrate introductions include sellers and buyers of exotic pets; acclimatization societies that import, establish, and relocate game animals and whose constituents include hunters and fishermen; and farmers and ranchers who import and experiment with novel lifestock (e.g., fitch farming). exotic vertebrates have in some instances great economic importance (as with lifestock and game animals), they also enjoy public popularity because of interest in hunting, fishing, eating, or viewing large and unusual animals in familiar environments. the negative ecological aspects of introduced vertebrates may be poorly understood by the public at large. such limited understanding may hinder control efforts and prevention of importation (bland & temple, ) . legislation has been passed in the united states to minimize risks of importing new and relocating existing vertebrate species. the lacey act passed in and ammended in was enacted to protect certain animals and endangered habitats, and to prevent introduction of noxious pests. under the act, violation of the law can result in fines and imprisonment [see usc w importation or shipment of injurious mammals, birds, fish (including mollusks and crustacea), amphibia, and reptiles; permits, specimens for museums; regulations m for more details]. similar legislation has been developed in new zealand. the biosecurity ( ) and hazardous substances and new organisms (hsno) ( ) acts were devised to protect the environment by preventing or managing the adverse effects of hazardous substances and exotic organisms. campbell ( ) points out that existing laws have many loopholes and are not effective when applied, indicating a need to improve existing regulations and to develop new laws to curtail unwanted entry by alien vertebrates. one proposal is to require importers of exotic organisms to develop "clean lists" and to prove that organisms are not potentially invasive and disrupting to native ecosystems (campbell, ) . legislative approaches limiting imports and exports of organisms may encounter complaints under the general agreement of tariffs and trade (gatt) that stricter quarantine measures are an unacceptable imposition of one country's environmental standards on others (camp-bell, ) . there is an obvious need for greater cooperation among interest groups, scientists, and legislators to devise solutions to problems associated with continuing introductions of exotic species and to provide direction for future action. as an evolutionarily stable control strategy development of resistance (behavioral or physiological) to pesticides (e.g., rodenticides) by vertebrates, and the need for repeated or multiple simultaneous control strategies (e.g., poisoning combined with trapping and hunting) indicate that control of vertebrates is an ongoing endeavor that attempts to reduce agricultural damage and losses (greaves, ) or to protect wilderness areas (cowan, ; morgan et al., ; payton et al., ) from pest damage. biological control has several advantages over chemical and cultural control practices (van driesche & bellows, ) : ( ) it is relatively cheap and biological control programs are often quicker to implement than to develop and to register new pesticides; ( ) use of carefully screened natural enemies increases selectivity of attack toward target pests; ( ) natural enemies in many instances are self-perpetuating and self-distributing; and ( ) development of resistance to natural enemies is extremely rare. one documented case of pests developing resistance to natural enemies is the development of resistance to myxomatosis by rabbits and corresponding attenuation of highly virulent strains of the myxoma virus to strains of intermediate virulence (fenner & ross, ) . the myxoma virusrabbit system in australia and europe is dynamic with increasing rabbit resistance selecting for more virulent strains of virus. this suggests that for the short-term, at least, the system is coupled in an antagonistic coevolutionary arms race (dwyer et al., ) . flexible natural-enemy behavior patterns and physiology have the potential to weaken evolutionary responses that can cause pest resistance to introduced control agents (holt & hochberg, ; jervis, ) . in comparison, pesticides and cultural controls tend to target a fixed physiological or behavioral function or pattern, and the resulting selection regime is constant allowing pests either to increase tolerance to poisons or to learn and develop avoidance behaviors (e.g., bait and trap shyness). spatial heterogeneity of natural-enemy attack limits selection pressure on hosts by natural enemies, thus reducing the rate of resistance development by pests compared with uniformly applied selection pressures such as pesticides. pests that escape attack move into enemy-free areas and continue breeding; thus, the rate of coevolution is reduced by susceptible pests in transient refuges (jervis, ) . at the metapopulation level, natural enemies may be ineffective selection agents because of widespread extinction and establishment of pest subpopulations that maintain pest susceptibility. additionally, resistance development may involve costs leading to a corresponding decrease in fitness. for example, increased tolerance to attack may reduce the pest's reproductive capacity and ability to compete for resources, or may increase susceptibility to other mortality agents (holt & hochberg, ) . there are opportunities to enhance biological control programs against vertebrate pests that cause social, agricultural, and conservation problems. in many instances, biological control offers the best chances for long-term control, particularly in isolated areas with rugged terrain, in suburban areas with high-density human populations, or in places where pests are nocturnal or secretive. biological control will not totally alleviate vertebrate pest problems. it may, however, reduce the vigor of pest populations, thereby reducing damage, minimizing nuisance value, or allowing native species to compete more effectively for food and breeding sites. programs could be initiated to simply reassociate host-specific micro-and macroparasites with pest populations that have depauperate natural-enemy faunas (dobson & may, ) , and there is no shortage of targets as small founding populations of vertebrates continue to invade and proliferate in new habitats. genetically engineered natural enemies are additional tools to aid biological control efforts. research with agents that cause immunocontraception will likely diversify as advances in molecular biology continue, and routes alternative to sterilization may be taken. this area of vertebrate biological control will be tested more thoroughly once small-scale and long-term field trials begin with sterilizing microorganisms. the effect of barn owls (tyto alba) on the activity and microhabitat selection of gerbillus allenbyi and g. pyramidum runaway rabbit 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history and biology of a successful colonizer the european rabbit, the history and biology of a successful colonizer the need to control cane toads comparative histopathological study of rabbit haemorrhagic disease (rhd) and european brown hare syndrome (ebhs) rabbit haemorrhagic disease in the united kingdom a propos d'un cas de capillariose a capillaria hepatica observe dans un elevage de lapins croises garenne the rabbit in new zealand the complete nucleotide sequence of rabbit haemorrhagic disease virus (czech stain c v ): use of the polymerase chain reaction to detect replication in australian vertebrates and analysis of viral population sequence variation resistance to anticoagulant rodenticides viral haemorrhagic disease or rabbits in mexico: epidemiology and viral characterization translocation and a species conservation tool: status and strategy when is biological control evolutionary stable (or is it analysis of vertebrate pest control biological control of vertebrate pests classical biocontrol: panacea or pandora's box? an evaluation of the biological control of the feral cat felis catus (linnaeus, ) and no birds sing: the story of ecological disaster in a tropical paradise rabbit and fox introductions in tierra del fuego: history and assessment of the attempts at biological control of the rabbit infestation parasitoids as limiting and selective factors: can biological control be evolutionary stable? biological control of possums long-term effects of porcine zonae pellucidae immunocontraception on ovarian function in feral horses (equus caballus) case studies in wildlife immunocontraception: wild and feral equids and white-tailed deer predation and population cycles of small mammals the natural regulation of animal numbers rabbit virus threatens ecology after leaping the fence capillaria hepatica infection: incidental finding in a dog with renal insufficiency european brown hare syndrome virus: molecular cloning and sequencing of the gonome a new viral disease in rabbits biological invasions: lessons for ecology comparative conservation biology of oceanic archipelagoes birth control for feral pests fluctuations of animal populations and a measure of community stability food webs: a plea for parasites thresholds and breakpoints in ecosystems with a multiplicity of stable states regulation and stability of hostparasite population interactions ii. destabilizing processes depression of host population abundance by direct life cycle macroparasites models to assess the potential of capillaria hepatica to control population outbreaks of house mice evaluation of a nematode (capillaria hepatica bancroft, ) as a control agent for populations of house mice (mus musculus domesticus schwartz and schwartz, ). revue scientifique et technique office international des epizooties quantifying the impact of disease on threatened species immunocontraception for wildlife population control detecting disease and parasite threats to endangered species and 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primer of conservation biology the effect of habitat on the helminth parasites of an island population of the polynesian rat (rattus exulans) testing the concept of virally vectored immunosterilization for the control of wild rabbit and fox populations in australia progress towards using a recombinant myxoma virus as a vector for fertility control in rabbits. reproduction, fertility and development the australian and new zealand calicivirus disease program the disappearance of guam's wildlife likely targets for immunocontraception in marsupials the establishment and spread of myxomatosis and its effect on rabbit populations escaped rabbit calicivirus highlights australia's chequered history of biological control a relationship between plagues of the house mouse mus musculus (rodentia: muridae) and prolonged periods of dry weather in south-eastern australia extinction of an island forest avifauna by an introduced snake regulation of mouse colony abundance by heligmosomoides polygyrus population dynamics in wild and laboratory rodents the role of parasites in regulating host abundance a harebrained scheme the potential of murine cytomegalovirus as a vital vector for immunocontraception. reproduction, fertility and reproduction immunological reactions and infertility can predators regulate small mammal populations? evidence from house mouse outbreaks in australia mammal populations: fluctuation, regulation, life history theory and their implications for conservation fertility control of mammal pests and the conservation of endangered marsupials effects of wild pig rooting in a deciduous forest the effects of capillaria hepatica (nematoda) on natality and survival to weaning in balb/c mice population dynamics of an outbreak of house mice (mus domesticus) in the mallee wheatlands of australia: hypothesis of plague formation the potential of capillaria hepatica to control mouse plagues the geographic distribution and host range of capillaria hepatica (bancroft) 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populations: fertility control of wildlife for conservation virus-vectored immunocontraception of feral mammals vermin and viruses: risks and benefits of viral-vectored immunosterilization changing social views of the desired degree of host range specificity of natural enemies of arthropods effects of feline panleucopaenia on the population characteristics of feral cats on marion island correlates of introduction in exotic new zealand birds when biotas meet: understanding biotic interchange science biological invasions and ecosystem processes: towards an integration of population and ecosystem studies the extent of biological control of rats with barn owls, tyto alba javanica in malaysian oil palm plantations. the planter biological control of vertebrate pest effectiveness and cost-efficiency of control of the wild rabbit, oryctolagus cuniculus (l.), by combinations of poisoning, ripping, fumigation, and maintenance fumigation response of wild rabbit populations to imposed sterility development and use of virus-vectored immunocontraception the characters of successful invaders the varying success of invaders biological control of vertebrates--a review, and an assessment of prospects for malaysia observations of the lifecycle of capillaria hepatica (bancroft, ) with a description of the adult acknowledgments i thank vincent d'amico, iii of bean's art ink for preparing the states geological survey provided information on the brown tree snake. key: cord- - kbq v w authors: heath, joan a.; zerr, danielle m. title: infections acquired in the nursery: epidemiology and control date: - - journal: infectious diseases of the fetus and newborn infant doi: . /b - - - / - sha: doc_id: cord_uid: kbq v w nan neonates, especially premature neonates, requiring intensive care support constitute a highly vulnerable population at extreme risk for nosocomial or health care-associated infections. it has been estimated that as many as % to % of infants who survive or more hours in a high-risk nursery or neonatal intensive care unit (nicu) acquire a nosocomial infection."* although nosocomial infections have long been recognized in nicus, only recently have data on rates been documented in the literature. as technology and treatments have advanced to significantly diminish mortality and morbidity among critically ill neonates, especially infants of very low birth weight (less than g), this vulnerability has only increased, as a result of both more profound immune system immaturity and more frequent use of invasive interventions that bypass skin and mucous membrane barriers. ' nosocomial infections in neonates carry high attendant morbidity and mortality and health care costs. prevention and control of these infections, although highly desirable, present a formidable challenge to health care professionals. because control over birth weight-the most significant predictor of nosocomial infection risk-is limited, proper nicu customs, environment, and procedures (e.g., hand hygiene, antimicrobial usage, catheter-related practices, skin and cord care, visitation policies, unit design, and staffing) can reduce the risk for infection in the nicu. understanding the epidemiology of nosocomial infections in neonates and methods for their prevention and control is critical to minimizing poor outcomes. this chapter describes the epidemiology, etiology, and clinical characteristics of neonatal nosocomial infections as well as the methods required for effective infection prevention and control. it is well recognized that the immune system of the newborn infant, especially the premature infant, is functionally inferior to that of older infants, children, and adults (see chapter ). the lineages of the cells that will develop into the immune system are present at the beginning of the second trimester. the major components of the neonatal immune system, including t cells, neutrophils, monocytes, and the complement pathways, are functionally impaired, however, when compared with those in older infants and adults. for example, neonatal neutrophils show decreased chemotaxis, diminished adherence to the endothelium, and impaired phagocyto~is~~~; neonatal complement levels and opsonic capacity also are reduced, particularly in the premature ne nate. '~ in addition, neonatal t cell lymphokine production, cytotoxicity, delayed-type hypersensitivity, and help for b cell differentiation all are inferior when measured against those in adults! antigenic naivete may account for many of these differences; however, inherent immaturity also appears to account for certain inequities. for example, neonatal t cells are delayed in their ability to generate antigen-specific memory function after hsv infection, even in comparison with naive adult t cells. ' passively acquired maternal immunoglobulin g (igg) is the sole source of neonatal igg. soon after birth, maternal igg levels begin to fall; weeks later, production of immunoglobulins by the neonate commences. neonatal igg levels reach about % of adult levels by year of age. unfortunately, because much of the maternal igg is not transferred to the infant until the last to weeks' gestation, premature infants start with significantly lower levels of serum igg than in their term counterparts, which persist throughout most of the first months of life. other issues specific to the premature neonate also affect the functional immune system. for instance, the immature gastrointestinal tract (lack of acidity worsened by use of histamine h blockers and continuous feedings) and easily damaged skin constitute open potential portals of entry for pathogens or commensals. in addition, like other intensive care unit populations, the nicu population frequently experiences extrinsic breeches of the immune system through use of intravascular catheters as well as other invasive equipment and procedures used to care for critically ill patients. it is generally accepted that colonization with "normal flora" prevents, to some degree, colonization by pathogenic organisms. the neonate begins life essentially sterile. in the healthy term neonate, colonization occurs within the first few days of life. the organisms involved by site are a-hemolytic streptococci in the upper respiratory tract, staphylococcus epidermidis and other coagulase-negative staphylococci (cons) on the skin, and gram-negative bacilli and anaerobes in the gastrointestinal tract. this process of colonization with normal flora is disrupted in infants cared for in an nicu in part because of exposure to the nicu environment, the hands of health care workers (hcws), antimicrobial agents, and invasive procedures. as a result, the microflora of infants in the nicu can be markedly different from that of healthy term infantses multiple antimicrobial agent-resistant cons, klebsiella, enterobacter, and citrobacter species colonize the skin and the respiratory and gastrointestinal tracts of a high proportion of nicu neonates by the second week of hospitali~ation.'~-'~ in addition, neonates in the nicu become colonized not only with candida albicans but also with non-albicans candida species and malasse~ia.'~-' ' because colonization of the neonate with pathogenic organisms is a prelude to invasive infection from the same pathogens? measures to prevent such colonization need to be considered. first, as a result of abnormal colonization, infants in the nicu themselves serve as an important reservoir of potential pathogens. second, contamination of the hands of hcws during routine patient care has been well documented.'* thus, careful attention to hand hygiene before and after contact with patients and their environment, as well as decontamination of potential fomites, are crucial measures in preventing spread of colonization and infection. nosocomial infections in healthy term infants are uncommon unless other conditions require that they be cared for in the nicu for several days to weeks. on the other hand, these other conditions are frequent in neonates of very low birth weight (less than g), who require prolonged nicu care. understanding the epidemiology of nosocomial infections in nicus can be challenging, because reported rates vary dramatically by institution. this variation probably results from use of nonstandard definitions of nosocomial infection and from differences in patient populations, such as mean gestational age, birth weight, and severity of underlying illness, which significantly affect the incidence of nosocomial infection." the national nosocomial infections surveillance (nnis) system is a national surveillance system of the centers for disease control and prevention (cdc) that uses standardized surveillance protocols and the involvement of multiple medical centers to provide benchmark data for the epidemiology of nosocomial nicu infections. using standardized definitions, "is reported in that , nosocomial infections occurred between and in , neonates in nicus.~' in this study, rates of intravascular catheterassociated bloodstream infection, the most frequent nosocomial infection, ranged from fewer than infections per umbilical or central catheter days in infants with a birth weight greater than g to almost infections per catheter days in the lowest-birth-weight group (less than another national, multicenter surveillance study, the pediatric prevention network's (ppn) point prevalence survey, was undertaken in to determine the point prevalence of nosocomial infections in nicus and to define risk factors associated with development of these infections." this study included infants from nicus. of the infants, ( . %) had an active nosocomial infection on the day of the survey. bacteremia accounted for % of infections; lower respiratory tract infections, ear-nose-throat infections, and urinary tract infections accounted for %, %, and %, respectively (table - ) . in contrast with the nicu setting, the frequency of nosocomial infection in well-baby nurseries has been estimated to be between . % and . %? - in general, non-lifethreatening infections such as conjunctivitis account for a majority of infections in the well-baby population. the remainder of this chapter focuses almost entirely on nosocomial infections in and control measures for the nicu setting. g). / ( . ) / ( ) / ( ) for purposes of surveillance and tracking, all infections occurring in hospitalized newborns could be considered nosocomial. infections that are manifested in the first few days of life, however, usually are caused by pathogens transmitted vertically from the maternal genital tract. unfortunately, no precise time point perfectly distinguishes maternally acquired neonatal infections from those transmitted within the nicu. nnis has attempted to address this issue by stratifying infections according to whether they are likely to be maternally acquired. in % of neonates who had an infection thought to be maternally acquired, onset occurred within hours of birth. use of a cutoff period of hours or less to designate maternally acquired infections allowed . % of bacteremias and . % of pneumonias to be considered as originating from a maternal source. maternally acquired bloodstream infections were more likely to be caused by group b streptococci, other streptococci, and escherichia coli, whereas those not maternally acquired usually were caused by coagulase-negative staphylococci. in general, nonmaternal routes of transmission of microorganisms to neonates are divided into three categories: contact (from either direct or indirect contact from an infected person or a contaminated source), droplet (from large respiratory droplets that fall out of the air at a maximum distance of feet), and airborne (from droplet nuclei, which can remain suspended in air for long periods and as a result travel longer distances). specific microorganisms can be spread by more than one mechanism; in most instances, however, a single mode of spread predominates. the cdc has developed a system of precautions for the control of nosocomial infections that is based on these modes of transmi~sion.~~ contact transmission of bacteria, viruses, and fungi on the hands of hcws is arguably the most important yet seemingly preventable means of transmission of nosocomial infection. spread of infection by this means can occur either by transmission of the hcw's own colonizing or infecting pathogens or, more often, by transmission of pathogens from one patient to another. that the hands of hcws become contaminated even in touching intact skin of patients has been well demonstrated.'* poor compliance with hand hygiene is another means by which the hands of hcws can spread organisms from one patient to another. furthermore, hands of hcws have been implicated in multiple outbreaks with a variety of different organisms; through experimental studies, a causal link between hand hygiene and nosocomial infection has been established. contact transmission by means of fomites also can occur and has been described as a potential mechanism of spread of pathogens in multiple nicu outbreaks. as described later in this chapter, implicated items have included linens, medical devices, soap dispensers, and breast pumps, to name a few. these observations highlight the need for careful attention to disinfecting items shared between infants. spread through large respiratory droplets is an important mode of transmission for pertussis and infections due to neisseria meningitidis, group a streptococci, and certain respiratory viruses, whereas airborne transmission by means of droplet nuclei is relevant for measles, varicella, and pulmonary tuberculosis. for large droplet or droplet nuclei transmission, usually an ill adult, either an hcw or a parent, is the source of infection in an nicu setting. in general, these organisms are rare sources of outbreaks. infusates, medications, and feeding powders or solutions can be intrinsically or extrinsically contaminated and have been reported as the source of outbreaks due to a variety of different pathogens. it is important when possible to mix infusates in a controlled environment (usually the pharmacy), to avoid multiuse sources of medication, and to use bottled or sterilized feeding solutions when breast milk is not available. of course, nosocomial infection also can arise from endogenous sources within the neonate. the "abnormal flora" of the neonate residing in the nicu, however, is determined at least in part by the nicu environment and hcws' hands. with use of molecular techniques, even organisms typically considered to originate solely from normal flora (e.g., cons) have been shown to have clonal spread in the hospital setting, suggesting transmission by means of the hands of hcws.~~,~' as discussed earlier, infants in nicus have intrinsic factors predisposing them to infection, such as an immature immune system and compromised skin or mucous membrane barriers. in addition, multiple extrinsic factors play important roles in the development of infection, such as presence of indwelling catheters, performance of invasive procedures, and administration of certain medications, such as steroids and antimicrobial agents. birth weight is one of the strongest predictors of risk for nosocomial infection. for instance, "is data demonstrate that compared with larger infants, low-birth-weight infants are at higher risk of developing bloodstream infections and ventilator-associated pneumonia, even after correction for central intravascular catheter and ventilator use." similarly, in the ppn's point prevalence survey, infants weighing g or less at birth were . ( % confidence interval [ci] . % to . %; p < . ) times more likely to have an infection than those weighing more than g. ' the relationship between birth weight and nosocomial infection is complicated by multiple other factors that accompany low birth weight and also increase risk for nosocomial infection. low birth weight, however, has been shown to be an independent predictor for nosocomial infection, after adjustment for use of vascular catheters, parented alimentation, and mechanical ~entilation.~~ it is likely that birth weight also is a surrogate marker for other unmeasured factors, such as immune system immaturity. central venous catheters (cvcs) increase the risk for development of nosocomial bloodstream infections. in a study by chien and colleagues , infants admitted to nicus in canada, nosocomial bloodstream infections were found to occur at a rate of . to . infections per catheter days, depending on the type of catheter, versus . infections per noncatheter days. other studies have demonstrated that the association between cvcs and bloodstream infection is independent of birth weight. ' mechanisms for cvc-related nosocomial bloodstream infections probably involve colonization of the catheter by means of the catheter hub, colonization of the skin at the insertion site? or hematogenous spread of pathogens from distant sites of infection or colonization. bloodstream infections also can result from contaminated intravenous fluids, which have the potential for intrinsic or, especially with use of multiuse vials, extrinsic contamination. factors related to the management of cvcs influence the risk of infection. disconnection of the cvc and the frequency of blood sampling through the catheter increase the frequency of catheter-related infection^.^^ by contrast, administration of a solution with heparin and exit-site antisepsis decreased infection. lower frequency of cvc tubing changes (every hours versus every hours) was associated with increased catheter contamination, suggesting a potential for increased risk of infecti n. ~ cvc management techniques, including use of antiseptic-impregnated dressings, antimicrobialcoated catheters, and avoidance of scheduled replacement of cvcs, are discussed in the most recent cdc recommendations, summarized in "guidelines for the prevention of intravascular catheter-related infection," published in and prepared by the hospital infection control practice advisory c~m m i t t e e .~~ it has been suggested that use of peripherally inserted central catheters (piccs) may be associated with a lower rate of infection than for other cvcs. studies based in nicus have yielded conflicting results. in a study by chien and colleagues?' the relative risk of bloodstream infection, after adjustment for differences in infant characteristics and admission illness severity, was . per catheter days for umbilical venous catheters, . for piccs, and . for broviac catheters, compared with no catheter ( p < . ). another study also documented similar rates of infection for broviac catheters and for piccs.~~ by contrast, a higher rate of infection with broviac catheters than with piccs was suggested by brodie and co-w~rkers.~~ further study of different cvcs in nicu infants is needed to delineate infection risks for individual catheter types. parented alimentation and intralipids have been shown to increase risk of bloodstream infection in premature infants even after adjustment for other covariables such as birth weight and cvc use. etiologic agents often associated are cons, candida species, and malassezia species. the pathogenesis of this association remains unclear. potential hypotheses are many. intralipids, for example, could have a direct effect on the immune system, perhaps through inhibition of interleukin- . alternatively, as with any intravenous fluids, parented alimentation has the potential for intrinsic and extrinsic contamination, and intralipids especially may serve as a growth medium for certain bacteria and fungi. finally, total parented alimentation and intralipids delay the normal development of gastrointestinal mucosa because of lack of enteral feeding, encouraging translocation of pathogens across the gastrointestinal mucosa. it is well accepted that mechanical ventilation is an important risk factor for nosocomial lower respiratory tract infection. a large multicenter study of neonates found that mechanical ventilation was a risk factor for bloodstream infection as well, even after adjustment for a number of covariables such as birth weight, parented nutrition, and umbilical catheterization:' clinically obvious respiratory infection appeared to precede some but not all cases of bloodstream infection associated with mechanical ventilation. the study authors suggested that the increased risk of mechanical ventilation could be attributed to colonization of humidified air, as well as to physical trauma from the endotracheal tube and its suctioning. a number of medications critical to the survival of infants in the nicu increase risk of infection. broad-spectrum antimicrobial agents, especially with prolonged use, are important in the development of colonization with pathogenic micro-organism~.~ the widespread use of broad-spectrum antimicrobial agents has been associated with increased colonization with resistant organisms in many settings, including nicus." in addition to colonization, antimicrobial agents also have been shown to increase risk of infection with resistant bacteria ' and with fungal pathogens!' other medications also appear to play a role in nosocomial infection. for instance, infants who receive corticosteroids after delivery are at approximately . to . times higher risk for nosocomial bacteremia in the subsequent to weeks than that observed for infants who do not receive this in addition, colonization and infection with bacterial and fungal pathogens have been shown to increase with the use of h, blocker^.'^"' measures of illness severity have been developed, in part, in an effort to account for variations in birth weight-adjusted mortality scores between nicus. the score for neonatal acute physiology (snap) was developed and validated by richardson and ass ciates, ~ and the clinical risk index for babies (crib) was developed by the international neonatal network.% these scores are highly predictive of neonatal mortality even within narrow birth weight strata and are predictive of nosocomial infection. thus, in investigating potential risk factors for nosocomial infection, it is important to consider adjusting for illness severity using such measures, in addition to adjusting for other potential confounders. other risk factors related to infection include poor hand hygiene and environmental issues, such as understaffing and overcrowding. b these and related issues are discussed later in this chapter under "prevention and control." nosocomial infections can affect any body site or organ system and manifest in a multitude of different ways. "is and ppn data demonstrated that bloodstream infections are the most common manifestation of nosocomial infection and account for % to % of infections (table - ; see also table - ). ,'' respiratory infections and eye, ear, nose, or throat infections are second and third in frequency, whereas gastrointestinal infections, urinary tract infections, surgical site infections, meningitis, cellulitis, omphalitis, septic arthritis, and osteomyelitis are reported less frequently."*" bloodstream infections are the most common and one of the most potentially serious nosocomial infections that occur in nicu patients. factors discussed earlier, including birth weight, intravascular catheters, mechanical ventilation, use of parented alimentation, and steroids, all have been shown to be associated with an increased risk of bloodstream infection. the most common pathogen associated with nosocomial bloodstream infections is cons (see table - ). staphylococcus aureus, enterococcus, candida species, e. coli, enterobacter species, klebsiella pneurnoniae, and pseudomonas aeruginosa also play important roles and are associated with higher morbidity and mortality rates than those associated with cons?^,^" in one study, the frequency of fulminant sepsis (fatal within hours) was estimated to be % ( % ci % to %) when the bloodstream infection was caused by pseudomonas species, whereas it was only % ( % ci % to %) when infection was caused by cons.^" the difficulty of assigning an etiologic role to cons on the basis of one blood culture that could be contaminated probably accounts for some distortion of the incidence and mortality data related to this organism, and this problem is discussed in detail in chapter . pneumonia accounts for % to % of nicu nosocomial infections" and has been associated with prolonged hospital stay and increased mortality. organisms most commonly associated with nosocomial pneumonia include cons, s. aureus, and i? aeruginosa (see table - ). mechanical ventilation and birth weight are important risk factors for nosocomial respiratory infection^.^^ diagnosis of nosocomial respiratory infections requires correlation of microbiologic results with clinical findings and can be challenging in lowbirth-weight infants because of the mostly nonspecific associated signs of illness and often misleading results of radiologic ~tudies.~' eye, ear, nose, and throat infections account for approximately % to % of infections, depending on birth weight." common etiologic organisms include cons and s. aureus, although gram-negative organisms, such as e. coli, e! aeruginosa, and k. pneumoniae, also can be isolated from these sites (see table - ). conjunctivitis appears to be the most common of these infections, accounting for % to %, depending on birth weight?' risk factors for neonatal conjunctivitis identified in a study from nigeria included vaginal delivery, asphyxia, and prolonged rupture of membranes. in the nnis review, gastrointestinal infections were estimated to account for % to % of nosocomial infections, depending on birth weight. necrotizing enterocolitis (nec) was the most common presentation.'" nec carries high morbidity and mortality rates. a review of nec epidemics estimated that surgery was required for a mean of % (range, % to %) of infants, and death occurred in a mean of % (range, % to y ) .~~ in controlled studies, identified risk factors for nec have included young chronologic age, low gestational age, low birth weight, and young age at first feeding. implication of specific pathogens is complex, requiring careful selection of an appropriate control population and attention to how and where specimens are collected. pathogens associated with nec outbreaks have included pseudornonas species, salmonella species, e. coli, k. pneumoniae, enterobacter cloacae, s. epidermidis, clostridium species, coronavirus, and r~t a v i r u s .~~'~~ the importance of infection control methods such as strict attention to hand hygiene and cohorting patients in the nicu is suggested by the observation that their implementation has been followed by resolution of the outbreak. a detailed discussion of the cause of nosocomial sepsis and meningitis is found in the chapter on bacterial sepsis (chapter ) and chapters describing specific etiologic agents. s. aureus is a colonizing agent in neonates and has been a cause of nosocomial infection and outbreaks in well-baby nurseries and nicus. methicillin-resistant s. aureus (mrsa) has become a serious nosocomial pathogen, and outbreaks have been reported in many areas of hospitals, including n~rseries.~~-~' in addition to the usual manifestations of neonatal nosocomial infection (conjunctivitis, bloodstream infections, and pneumonia), nosocomial s. aureus infections can manifest as skin infection^?^ bone and joint infections,@' parotitis:' staphylococcal scalded skin syndr me, ~*~~ toxic shock syndrome? and disseminated sepsis. the role of the hands of hcws in transmitting and spreading pathogenic organisms among infants was demonstrated with s. aureus in the ~."*~~ currently, in a majority of instances, s. aureus transmission is thought to occur by direct contact. thus, it is not surprising that understaffing and overcrowding have been associated with s. aureus outbreaks in n i c u s .~~.~ the potential for airborne transmission, however, has been suggested by the occurrence of "cloud babies? described by eichenwald and colleagues in . "cloud" hcws also have been described; in such cases, the point source of an outbreak was determined to be a colonized hcw with a viral respiratory infe~tion.~~'~' in one of these studies, dispersion of s. aureus from the implicated hcw was found to be much higher after experimental infection with rhinovirus. more recently, molecular techniques not only have defined outbreaks but also have demonstrated that transmission to infants probably occurs from colonized hcws, * and sometimes from colonized parents. nasal mupirocin ointment has been used to control outbreaks of both methidin-susceptible s. aureus and mrsa. , the pharynx, rather than the anterior nares, however, may be a more common site of colonization in neonates and infants," and eradication of the causative organisms with nasal mupirocin may be more difficult in this site. since the early s, cons has been the most common cause of nosocomial infection in the nicu. ' this finding suggests that a portion of cons infections may be preventable by strict adherence to infection control practices. the fact that a hand hygiene campaign was associated with increased hand hygiene compliance and a lower rate of cons-positive cultures supports this ~ontention.'~ enterococcus has been shown to account for % of total nosocomial infections in neonates, % to % of bloodstream infections, % to % of cases of pneumonia, % of urinary tract infections, and % of surgical site sepsis and meningitis are common manifestations of enterococcal infection during nicu outbreak^'^,^^; however, polymicrobial bacteremia and nec frequently accompany enterococcal sepsis. identified risk factors for enterococcal sepsis, after adjustment for birth weight, include use of a nonumbilical cvc, prolonged presence of a cvc, and bowel resection?' because enterococcus colonizes the gastrointestinal tract and can survive for long periods of time on inanimate surfaces, the patient's environment may become contaminated and, along with the infant, serve as a reservoir for ongoing spread of the organism. the emergence of vancomycin-resistant enterococci (vre) is a concern in all hospital settings, and vre have been the cause of at least one outbreak in the nicu setting ' in the neonate, resistant strains appear to cause clinical syndromes indistinguishable from those due to susceptible enteroco~ci.'~ the conditions promoting vre infection, such as severe underlying disease and use of broad-spectrum antimicrobial agents, especially vancomycin, can be difficult to alter in many nicu settings. guidelines for the prevention and control of vre infection have been published; these focus on infection control tools such as rapid identification of a vrecolonized or vre-infected patient, cohorting, isolation, and barrier precautions. historically, before the recognized importance of hand hygiene and the availability of antimicrobial agents, group a streptococci (gas) were a major cause of puerperal sepsis and fatal neonatal sepsis. although less common now, gas continue to be a cause of well-baby and nicu outbreak^.'^-'^ gas-associated clinical manifestations include severe sepsis and soft tissue infections. one report described a high frequency of "indolent omphalitis"; in this outbreak, the umbilical stump appeared to be an important site of gas colonization and an ongoing reservoir of the organism." routine cord care included daily alcohol application. after multiple attempts, the outbreak finally was interrupted after a -day interval during which bacitracin ointment was applied to the umbilical stump in all infants, and affected infants received intramuscular penicillin. molecular techniques have enhanced the ability to define outbreaks, and use of these techniques has suggested that transmission can occur between mother and infant, between hcw and infant, and between infantsprobably indirectly on the hands of hcws.",~~ in one recurring outbreak, inadequate laundry practices appeared to be a contributing factor. nnis data have shown that group b streptococci (gbs) infections account for less than % of non-maternally acquired nosocomial bloodstream and pneumonia infections." a number of studies from the s and s demonstrated nosocomial colonization of infants born to gbs-negative ~o m e n .~~-~o these studies suggested a rate of transmission to babies born to seronegative mothers as high as % to / . ~,~' a recent case-control study evaluating risk factors for lateonset gbs infection demonstrated that premature birth was a strong predictor?' in that study, % of the infants with late-onset gbs infection were born at less than weeks of gestation (compared with % of controls), and only % of the mothers of these infants were colonized with gbs, suggesting possible nosocomial transmission of gbs during the nicu stay. the hands of hcws are assumed to account for the transmission of most cases of nosocomial gbs infection. breast milk also has been implicated as a potential mode of acquisition, however. in one report, gbs probably was transmitted from breast milk to one set of premature triplets between days and of life. two maternal vaginal swabs taken before delivery did not grow gbs, but repeated cultures of the mother's breast milk yielded a pure growth of gbs (greater than ' colony-forming units [ cfu] /ml) despite no evidence of mastitis. in this report, antimicrobial therapy administered to the mother appeared to eradicate the organism. the enterobacteriaciae family has long been recognized as an important cause of nosocomial infection. neonatal infection can be manifested as sepsis, pneumonia, urinary tract infections, and soft tissue infections; morbidity and mortality rates frequently are enterobacter species, k. pneumoniae, e. coli, and serratia marcescens are the members of the family enterobacteriaciae most commonly encountered in the nicu. enterobacter species have been estimated to account for % of bloodstream infections, % of cases of pneumonia, and % of surgical site infections in the nicu setting (see table - ). outbreaks due to enterobacter species in nicus have been associated with thermometer^?^ a multidose vial of d e x t r o~e~~ intravenous fluids,% and powdered formula? as well as with understaffing, overcrowding, and poor hand hygiene practice^.'^ in one outbreak in which contaminated saline was linked to the initial cases, subsequent ongoing transmission was documented, presumably by means of the hands of hcws and the environment." in that study, early gestational age, low birth weight, exposure to personnel with contaminated hands, and e. cloacae colonization of the stool were associated with e. cloacae bacteremia, whereas use of cvcs and mechanical ventilation was not. k. pneumoniae has been estimated to account for a similar proportion of infections in the nicu setting to that identified for enterobacter species. investigations in outbreaks involving klebsiella species have implicated contaminated breast milk, oo infusion therapy practices,"' intravenous dextrose,io cockroaches, di~infectant,"~ incubator humidifier^,'^' thermometers, oxygen saturation probes,io and ultrasonography coupling ge .io in a surveillance study of nicu infants in brazil, % became colonized with kleb~iella.'~ in this study, colonization was associated with use of a cephalosporin and aminoglycoside combination therapy, as well as with longer duration of the nicu stay. e. coli has been estimated to cause % of bloodstream, % of gastrointestinal, and % of surgical site infections. e. coli also has been responsible for outbreaks of pyelonephritis,io ga~troenteritis,'~'*"o and nec. s. marcescens is an opportunistic pathogen that survives in relatively harsh environments. disease due to s. marcescens often is manifested as meningitis, bacteremia, and pneumonia.'" s. marcescens infections have a high potential for morbidity and mortality." ,' s. marcescens outbreaks have been associated with, but not limited to, contaminated soap, multiuse bottles of theophylline," formula,"' enteral feeding additives,l breast pumps,' ",'i and transducers from internal monitors.ii although point source environmental contamination is important in serratia outbreaks, in many of these outbreaks and in reports in which no point source was identified,"' patient-to-patient spread of the organism by means of the hands of hcws appeared to be an important mechanism of spread.i extended-spectrum p-lactamases (esbls) are plasmidmediated resistance factors produced by members of the enterobacteriaceae family. esbls inactivate third-generation cephalosporins and aztreonam. they most commonly occur in k. pneumoniae and e. coli but have increasingly been found in other gram-negative bacilli. colonization with esblproducing organisms has been associated with administration of certain antimicrobials and longer duration of hospitalization, whereas infection has been associated with prior colonization and use of cvcs.i that the esbl-containing plasmids can be transmitted to other enterobacteriaceae organisms has been demonstrated in nicu outbreaks in which the implicated plasmid spread from klebsiella species to e. coli, e. cloacae, and citrobacter fieundii.' s'zl the gastrointestinal tract in neonates and the hands of hcws serve as reservoirs for members of the enterobacteriaceae family. thus, in general, measures aimed at controlling spread of organisms in this family have focused on attention on hand hygiene, cohorting of patient and staff, and observation of isolation precautions.' " i? aeruginosa, an opportunistic pathogen that persists in relatively harsh environments, frequently has been associated with nosocomial infections and outbreaks in the nicu setting. nosocomial i! aeruginosa infections vary in their clinical presentation, but the most common manifestations are respiratory, ear, nose, or throat and bloodstream infections.*' from the ppn data it has been estimated that j? aeruginosa species account for . % of total pathogens, % of bloodstream infections, and % of respiratory infections. l ? aeruginosa infections, particularly bloodstream infections, have been associated with a very high mortality rate.' feeding intolerance, prolonged parented alimentation, and long-term intravenous antimicrobial therapy have been identified as risk factors for pseudomonas infe~ti n.l~~ outbreaks due to i! aeruginosa have been linked with contaminated hand lotion,' respiratory therapy solution, ' a water bath used to thaw fkesh-frozen plasma,' a blood gas analy~er,'~' and bathing sources. in one case, neonatal pseudomonas sepsis and meningitis were shown by pulsed-field gel electrophoresis to be associated with shower tubing from a tub used by the infant's mother during labor.i ' of importance, hcws and their contaminated hands also have been linked with pseudomonas infections in the nicu setting. in a study of a new york outbreak, recovery of pseudomonas from the hands of hcws was associated with older age and history of use of artificial nails. ' this and other studies suggest that the risk of transmission of pseudomonas to patients is higher among hcws with onychmycosis or those who wear long artificial or long natural nail^.'^^,'^^ as a result of these and other findings, the cdc revised its hand hygiene recommendations to include a recommendation against the presence of hcws with artificial fingernails in intensive care units.' bordetella pertussis is a rare cause of nosocomial infection in neonates. when b. pertussis infection occurs, parents and hcws typically are the source. a parent was the source of an outbreak involving three neonates and one nurse in a special care nursery in a~stralia.'~' in in knoxville, tennessee, an outbreak involving six neonates probably was due to transmission of infection by an hcw."' as a result of the tennessee outbreak, infants received erythromycin prophylaxis. subsequently, an increase in infantile hypertrophic pyloric stenosis was noted by local pediatric surgeons. results of a cdc investigation suggested a causal role of erythromycin in the cases of hypertrophic pyloric steno~is.'~~~~" erythromycin remains the recommended agent of choice for prophylaxis after pertussis exposure, but parents should be informed of the risk and signs of hypertrophic pyloric stenosis, and cases associated with erythromycin use should be reported to medwat~h.'~~ newborn infants are particularly prone to infection and disease following exposure to mycobacteriurn tuberculosis. a cluster of multidrug-resistant m. tuberculosis infections was noted in three infants born during a -week period in one new york h spita .l~~ investigation implicated an hcw who visited the nursery several times during that period. pulmonary and extrapulmonary disease occurred in three infants to months after exposure, highlighting the vulnerability of the newborn p~pulation.'~~ tuberculosis screening of hcws, ultraviolet lighting, and a high number of air exchanges appear to be effective methods in preventing nosocomial tuberculosis infe~ti n.i~' the cdc's "guidelines for preventing the transmission of mycobacteriurn tuberculosis in health-care settings" emphasizes ( ) use of engineering controls and personal protective equipment, ( ) risk assessments for the development of institutional tuberculosis control plans, ( ) early identification and management of individuals with tuberculosis infection and disease, ( ) tuberculosis screening programs for hcws, ( ) hcw education and training, and ( ) evaluation of tuberculosis control prograrns.l * candida species are an increasingly important cause of nosocomial infection in nicu patients and have been estimated to account for . % of bloodstream infections and % of urinary tract infection^.^^^"^ prospective studies have estimated colonization rates with candida to be % to % in low-birth-weight neonate^,'^"^^-'^' and colonization has been associated with subsequent invasive disease. ' the mortality rate can be high in invasive candidiasis. in one study of patients with fungemia due to candida species, a case-fatality rate of % was r e~ r t e d . i~~ risk factors for fungal infections in neonates are similar to risk factors for bacterial infections; low birth weight and gestational age are important predictors. in addition, a prospective, multicenter study of infants found that use of a third-generation cephalosporin, presence of a cvc, intravenously administered lipids, and hz blocker therapy were associated with candida colonization after adjusting for length of stay, birth weight of g or less, and gestational age less than weeksl candida parapsilosis appears to be the most frequent species associated with nosocomial candida infection in nicu infants. both cross-contamination and maternal reservoirs are sources of nosocomial candida albicans infection, as demonstrated in studies using molecular typing method^.'^-'^^ malassezia species, lipophilic yeasts, frequently colonize nicu patients. in one french study, of preterm neonates ( %) became colonized with malassezia fit+r. malassezia pachydermatis, a zoonotic organism present on the skin and in the ear canals of healthy dogs and cats, also has been associated with nosocomial outbreaks in the nicu setting. , in one report, the outbreak appeared to be linked to colonization of hcws' pet dogs.i ' pichia anornala, or hansenula anornala, a yeast found in soil and pigeon droppings, and on plants and fruits, also can colonize the human throat and gastrointestinal tract. in general, it is an unusual cause of nosocomial infection in neonates, but it was the cause of two reported outbreaks in this ~e t t i n g . '~~, '~~ in both reports, carriage on the hands of hcws appeared to be a factor. invasive mold infections are a rare cause of nosocomial infection in neonates, but when they occur, they are associated with high mortality rate. aspergillus infections may manifest as pulmonary, central nervous system, gastrointestinal, or disseminated disease. a cutaneous presentation, with or without subsequent dissemination, appears to be the most common presentation for hospitalized premature infants without underlying immune defi~iency.'~'.'~~ often, skin maceration is the presumed portal of entry. in a series of four patients who died of disseminated aspergillus infection that started cutaneously, a contaminated device used to collect urine from the male infants was impli~ated.'~' similarly, contaminated wooden tongue depressors, used as splints for intravenous and arterial cannulation sites, were associated with cutaneous infection due to rhizopus microsporus in four premature infants.' in addition to preterm birth, use of broad-spectrum antimicrobial agents, steroid therapy, and hyperglycemia are thought to be risk factors for mold infection. even zoophilic dermatophytes have been described as a source of nosocomial infection in neonates. in one report, five neonatal cases in one unit were traced to an infected nurse and her cat. prolonged therapy for both the nurse and her cat was necessary to clear their infections. although many pathogens can cause nosocomial gastroenteritis, rotavirus is responsible for % or more of viral infections in high-risk nurseries, including the nicu." in one longitudinal study, rotavirus infection developed during hospitalization in of neonates ( ? ).'~~ in this study, rotavirus was manifested as frequent and watery stools in term infants and as abdominal distention and bloody, mucoid stools in the preterm neonates. a high titer of virus is excreted in stool of infected persons, and the organism is viable on hands and in the environment for relatively prolonged periods of time.' " attention to hand hygiene and disinfection of potential fomites are crucial in preventing spread of infection. this concept is illustrated by the results of one study in which rotavirus infection was associated with ungloved nasogastric tube feeding. respiratory viruses including influenza a virus, parainfl uenza virus, coronavirus, respiratory syncytial virus, and aden )virus have been reported to cause nosocomial infections in qicu patient^.'^^-'^' associated clinical findings include rhino rrhea, tachypnea, retractions, nasal flaring, rales, and wheezir g, but illness also can be manifested as apnea, sepsis-like i lness, and gastrointestinal symptoms. ', * identified risk f ictors for acquisition vary from study to study but have includl id low birth weight, low gestational age, twin pregnancy, mech anical ventilation, and high crib s~o r e . '~~-'~* contact and d :oplet transmission are the most common modes of sprcad of infection, again highlighting the importance of scrul (dous hand hygiene in delivery of patient care for this popul, tion. numerous nursery and nicu outbreaks of enterovii a infection have been r e p~r t e d . '~~"~ in the neonate with e iteroviral infection, clinical manifestations can range fron mild gastroenteritis to a severe and fulminant sepsis-like sync home or meningitis/encephalitis. the latter presentation c m be associated with a high mortality rate.lw in index cas :s, the patient may have acquired disease vertically, with subst quent horizontal spread leading to outbreak^'^^'^^; with othe r viral pathogens, virus can be shed into the stool for prolnged periods, enabling patient-to-patient transmission by the hands of hcws when hand hygiene procedures are impr iperly performed. congenitally acquired cytomegalovirus (cmv) infecti )n is a cause of morbidity and occasionally death, whereas postnatally acquired cmv infection follows a benign col rse in virtually all healthy term infants. postnatal cmv infi ction, however, can cause considerable morbidity and death i n premature infants. hepatitis, neutropenia, thrombocyto penia, sepsis-like syndrome, pneumonitis, and developmi nt of chronic lung disease each have been associated with postnatal acquisition of cmv in premature infant^.'^^''^^ w th the routine use of cmv-seronegative blood products in these neonates, a majority of postnatal cmv infections ap ear to be acquired through breast milk.' it has been estinated that transmission by this mode occurs in approximate y % of breast-fed infants of mothers with cmv detec ed in breast milk. ' in one study, approximately % of these infants had clinical features of infection, and % pre iented with a sepsis-like syndrome. nosocomial person-to-] ierson transmission has been d o~u m e n t e d , '~'~'~~ but the exl ent to which this occurs is contr~versial.'~~ at present, no p :oven, highly effective method is available for removing cml ' from breast milk without destroying its beneficial compc nents. some data, however, suggest that freezing the breas: milk before use may decrease the cmv titer, thereby liniting subsequent transmission."* in a majority of cases, neonatal herpes simplex virus hsv) infection is acquired vertically from the mother. nursery transmission of hsv infection is rare but has been describe( . [ ] [ ] [ ] in each of these cases, hsv- was involved. in one infa it, the source of virus was thought to be a patient's father, wl had active herpes labiali~.'~~ subsequent spread of virus from this first infant to a second infant was thought to have occurred by means of the hands of an hcw. in another report, the source of hsv for the index case, an infant who died of respiratory distress in whom evidence of hsv infection was found at postmortem examination of the brain, was un- the hands of hcws were implicated in the spread of hsv to three subsequent cases, however. in another report, direct spread from an hcw was thought to be responsible for transmission of hsv to three infants over a period of approximately years.' studies of adults with herpes labialis suggest a high frequency of recovery of virus from the mouth and the hands ( % and %, re~pectively).'~~ in this same study, hsv was shown to survive for to hours on skin, cloth, and plastic. implementing contact precautions for infants with hsv and instructing hcws with active herpes labialis regarding control measures, such as covering the lesion, not touching the lesion, and using strict hand hygiene, are reasonable means to prevent nosocomial transmission of hsv. if there are concerns that an hcw would be unable to comply with control measures or if the hcw has a herpetic whitlow, such persons should be restricted from patient contact. nosocomial transmission of varicella in the nicu setting, although unusual, has been de~cribed.'~~ large-scale outbreaks in nurseries and nicus are rare, most probably because of the high rate of varicella-zoster virus (vzv) immunity in hcws and pregnant women. premature infants born at less than weeks of gestation are unlikely to have received protective levels of vzv igg from their mothers, so their potential risk is significant if an exposure occurs. transmission is most likely to occur from an adult with early, unrecognized symptoms of varicella. in such instances, the potential risk for vzv-seronegative exposed infants and hcws is substantial, especially if the patient in the index case is an hcw.'" for this reason, it is recommended that hcws be screened for prior varicella infection by history, with subsequent immunization as indicated. hepatitis a is a rare cause of nosocomial infection in nicus, but a number of outbreaks in this setting have been r e p~r t e d . '~' . '~~ in most instances, disease in neonates is clinically silent. neonatal cases often are detected only through recognition of the symptomatic secondary adult cases. in one report, disease was acquired by patients in the index cases through blood transfusion from a donor with acute hepatitis of note, the virus subsequently spread to another infants, nurses, and other hcws. overall, hepatitis a affected % of the patients and % of the nurses. lapses in infection control practices and the prolonged shedding of the virus in infants stool probably contributed to the rapid spread and high attack rate documented in the outbreak. outbreaks such as this one are unlikely because of current blood product practices to eliminate transmissible agents from donor blood. an effective infection control program that focuses on reducing risk on a prospective basis can decrease the incidence of nosocomial infection^.'^"^^ the principal function of such a program is to protect the infant and the hcw from risk of hospital-acquired infection in a manner that is cost-effective. activities crucial to achieving and maintaining this goal include collection and management of critical data relating to surveillance for nosocomial infection, and direct intervention to interrupt the transmission of infectious diseases. reducing the incidence of nosocomial infection for neonates must begin with surveillance for these events. surveillance has been defined as "a comprehensive method of measuring outcomes and related processes of care, analyzing the data, and providing information to members of the health care team to assist in improving those outcomes."' essential elements of a surveillance program include the following: defining the population and data elements as concisely collecting relevant data using systematic methods consolidating and tabulating data to facilitate evaluation analyzing and interpreting data reporting data to those who can bring about changeia surveillance systems necessarily vary, depending on the population; accordingly, a written plan, based on sound epidemiologic principles,ls should be in place to track rates of infection over time. because new risks can emerge, such as new interventional technology or drugs, changing patient demographics, and new pathogens and resistance patterns, the plan should be reviewed and updated the joint commission on accreditation of healthcare organizations (jcaho) recommends that hospitals have a written infection control plan that includes a description of prioritized risks; a statement of the goals of the infection control program; a description of the hospital's strategies to minimize, reduce, or eliminate the prioritized risks; and a description of how the strategies will be evaluated. the jcaho further recommends that hospitals identify risks for transmission and acquisition of infectious agents (table - system provides high-risk nursery-specific data collection methods as well as denominator data and allows external benchmarking of infection rates for this populati~n.'~~,'~' the nnis system defines a nosocomial infection as a localized or systemic process that results from adverse reaction to the presence of an infectious agent(s) or its toxin(s) and that was not present or incubating at the time of admission to the hospital. nnis also recognizes as special situations, and defines as nosocomial, some infections in neonates that result from passage through the birth canal but do not become clinically apparent until several or more days after birth. it does not, however, consider infections that are known or proved to have been acquired transplacentally to be noso-~omial.'~' distinction between maternal and hospital sources of infection is important, although difficult at times, because control measures designed to prevent acquisition from hospital sources will be ineffective in preventing perinatal acquisition of pathogen^.'^' surveillance for infections in healthy newborns also is challenging because of the typically short length of stay. infections can develop after discharge, and these are more difficult for infection control practitioners (icps) to capture. methods for postdischarge surveillance have been developed, but because most neonatal infections that occur following discharge are noninva~ive,'~~ such surveillance has not been widely implemented, because of concerns about the cost-effectiveness of these labor-intensive processes. the ultimate goal of surveillance is to achieve outcome objectives (e.g., decreases in infection rates, morbidity, mortality, or cost).is baseline infection rates for an inpatient unit must be established so that the endemic rate of infection can be understood and addressed. in the nicu, concurrent surveillance (initiated while the infant is in the hospital) should be conducted by persons trained to collect and interpret clinical information. typically, such persons are icps working closely with hcws and using various data sources (table - ) . using nnis or other accepted definitions, the icp should collect data regarding cases of nosocomial infection in the nicu population as well as population-specific denominator data. denominators must be carefully chosen to represent the population at risk. attempts to stratify risk should take into account both underlying infant-specific risks and those resulting from therapeutic or diagnostic interventions.' risk stratification techniques that attempt to control for distribution of risk have included severity of illness score, intensity of care required, and birth weight. because the risk for developing nosocomial infection is greater for lower-birthweight infants:' the nnis system breaks down data collection and analysis into birth weight categories (tables - and - ).' ' the use of invasive devices, however, also is an important factor to consider. the appropriate denominator for an infection related to the use of a medical device, such as a cvc-related primary bloodstream infection, according to nnis, would be total device days for the population during the surveillance period. the formula generally used for calculating nosocomial infection rates is (x/y)k, where x equals the number of events (infections) over a specific time period, y equals the population at risk for development of the outcome, and k is a constant and a multiple of . rates can be expressed as a percentage (k = loo), although device-related infections usually are expressed as events per device days (k = ). a value should be selected for k that results in a rate greater than because use of invasive devices is such a significant risk factor both for bloodstream infection and ventilatorassociated pneumonia, assessing nicu practices with device use may be warranted. "is provides a benchmark for nicu device utilization broken down into birth weight categories. an nicu device utilization ratio can be calculated using the following formula: in those units with device utilization ratios above the nnis th percentile, investigation into the practices surrounding use of invasive devices may be ~a r r a n t e d . '~~ calculating monthly and annual rates to employ as benchmarks can assist in identification of a potential problem in device-related procedures. surveillance data must be arranged and presented in a way that facilitates interpretation, comparison both directed internally and with comparable external benchmarks, and dissemination within the organization. quality improvement tools (e.g., control and run charts) can be useful for these purposes. statistical tools should be used to determine the significance of findings, although statistical significance should always be balanced with the evaluation of clinical ~ignificance."~ external benchmarking through interhospital comparison is a valuable tool for improving quality of are'^,'^^ but should be performed only when surveillance methodologies (e.g., case definitions, case finding, data collection methods, intensity of ~urveillance)'~~ can reasonably be assumed to be consistent between facilities. few overall infection rates in nicus are available, but a small study done in children's hospitals performing nicu nosocomial infection surveillance reported a median nosocomial infection rate of . infections per patient days (range, . to . ). ' nnis does not provide a benchmark for overall infection rates within nicus. instead, nnis provides birth weight-stratified device-associated infection rates for umbilical and central intravascular line-associated bloodstream infections. the most recent rates for catheterrelated bloodstream infections ( to nicus reporting) and ventilator-associated pneumonias ( to nicus reporting) are summarized in table e~ . l~~ once arranged and interpreted, nosocomial infection data must be shared with personnel who can effect change and implement infection control interventions. written reports summarizing the data and appropriate control charts should be provided to the facility's infection control committee, unit leaders, and members of the hospital administration on an ongoing basis. the interval between reports is determined by the needs of the institution. in addition to formal written reports, face-to-face reports are appropriate in the event of identification of a serious problem or an outbreak. icps can serve as consultants to assist nicu or neonatology service leaders in addressing infection rate increases or outbreak management. surveillance activities typically identify endemic nosocomial infections (i.e., those infections that represent the usual level of disease within the nursery or nlcu).' although the rate can fluctuate over time, in the absence of interventions that successfully reduce risk of infection, the difference rarely is statistically significant. establishing an nicu's endemic infection rate and expected variation around that rate allows the icp to rapidly identify unusual increases in rates that may indicate on outbreak (epidemic) of a particular infection. using baseline surveillance data along with aggregate data from sources such as the "is system allows the icp to develop meaningful threshold rates for initiating outbreak investigation.is alternatively, hcws can be the first to sense an increase in infections, which then can be confirmed or refuted by surveillance data? even a single case of infection due to an unusual and potentially dangerous pathogen (e.g., salmonella) can constitute the index case for a subsequent outbreak and thus merits rapid and comprehensive investigation. outbreaks may need to be reported to health authorities, depending on local and state requirements as well as the organism involved. numerous studies have described nursery and nicu epidemics caused by a variety of pathogens (table - ) , and most such epidemics have required the coordinated efforts of icps, nicu leadership, staff, and hospital administration outbreak investigation and intervention should be approached systematically, applying sound epidemiologic principles. in general, the process should include the follo~ing'~~~~''": for resolution. , , , , i j i, ,zo - confirming that an outbreak exists, by comparing the outbreak infection rate with baseline data (or with rates reported in the literature if baseline data are not available), and communicating concerns to stakeholders within the institution (and to those in other agencies if notification of health authorities is necessary) assembling the appropriate personnel to assist in developing a case definition and in planning immediate measures to prevent new cases performing active surveillance using the case definition to search for additional infections and collecting critical data and specimens characterizing cases of infection by person, place, and time, including plotting of an epidemic curve (to facilitate identification of shared risk factors among involved patients, such as invasive devices, proximity to other infected patients or temporal association with infection in such patients, common underlying diagnoses, shared medical or nursing staff, surgery, and medications, including antimicrobial agents) formulating a working hypothesis and testing this hypothesis (if the severity of the problem warrants this level of study, and provided that the institution has and can commit the necessary resources), with use of analytic approaches, including case-control and cohort studies, as appropriate to determine the likely cause of the outbreak instituting and evaluating control measures, which can be implemented anywhere in the foregoing process (more directed measures become possible as more is learned about the outbreak, and efficacy of control measures can be judged on whether the outbreak resolves, as indicated by return of number of cases to endemic levels or by cessation of occurrence of infections) reporting findings to appropriate personnel, including unit staff, hospital administration, and public health authorities (if involved in management of the outbreak), in comprehensive written reports, including summaries of how the outbreak was first recognized, study and analysis methods used, interventions implemented to resolve the epidemic, results, and a discussion of any other important outcomes or surveillance and control measures identified interventions used to control and limit outbreaks usually have consisted of isolation and cohorting of infected or colonized infants to prevent transmission of organisms. transmission-based precautions, a system developed by the cdc, can be used to determine the most effective barrier precautions to use with affected patients. cohorting, or placing infants infected or colonized with the outbreak organism together in geographically segregated areas and assigning dedicated staff and equipment to their care, also has been used successfully to halt outbreaks in nurseries and nicus. in extreme cases, closure of an nicu to admissions has been necessary to bring an outbreak under ~o n t r o l .~~~'~~, '~~ every attempt should be made to identify the source of a nursery outbreak, although this is not always possible. sources implicated in nicu outbreaks have included medications, equipment, and enteral feeding solutions; person-to-person transmission and environmental reservoirs also have been efforts to identify the source may include culturing of specimens from hcws, equipment, and the environment, although careful consideration should be given to the potential benefits before initiating these measures. culture of samples from the environment and equipment, in view of the vast number of objects that could be contaminated, usually is not helpful in identifying the source of an outbreak unless specific case characteristics or microbiologic data strongly suggest the location. culture of specimens obtained from hcws when person-to-person transmission is suspected may be more likely to identify the source of an outbreak, but it must be remembered that an hcw whose culture specimen yields the outbreak organism may have been transiently colonized while working with an affected infant, rather than constituting the source of the infection. management of culture-positive hcws (possible furlough, treatment, and return to work criteria) should be planned in advance of widespread culture surveillance and should involve supervisors of affected employees and occupational health services.'" reported. , , , , , the most widely accepted guideline for preventing the transmission of infections in hospitals was developed by the cdc. most recently revised in , the system contains two tiers of precautions. the first and most important, standard precautions, was designed for the management of all hospitalized patients regardless of their diagnosis or presumed infection status. the second, transmission-based precautions, is intended for patients documented or suspected to be infected or colonized with highly transmissible or epidemiologically important pathogens for which additional precautions to interrupt transmission are needed. standard precautions are designed to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources and are to be followed for the care of all patients, including neonates. they apply to blood; all body fluids, secretions, and excretions except sweat; nonintact skin; and mucous membranes. components of standard precautions include hand hygiene and wearing gloves, gowns, and masks and other forms of eye protection. hand hygiene plays a key role for caregivers in the reduction of nosocomial infection for patient^'^.^'^ and in prevention of nosocomial or health cart+associated infections. hand hygiene should be performed before and after all patient contacts; before donning sterile gloves to perform an invasive procedure; after contact with blood, body fluids or excretions, mucous membranes, nonintact skin, and wound dressings; in moving from a contaminated body site to a clean body site during patient care (ie., from changing a diaper to performing mouth care); after contact with inanimate objects in the immediate vicinity of the patient; after removing gloves; and before eating and after using the re~troom.'~~ when hands are visibly soiled or contaminated with proteinaceous materials, blood, or body fluids, and after using the restroom, hands should be washed with antimicrobial soap and water. soaps containing % to % chlorhexidine gluconate or . % t r i~l o s a n '~~ are recommended for hand washing in n~rseries.'~~ when hands are not visibly soiled, alcohol-based hand rubs, foams, or gels are an important tool for hand hygiene. compared with washing with soap and water, use of the alcohol-based products is at least as effective against a variety of pathogens and requires less time, and these agents are less damaging to skin. the cdc "guideline for hand hygiene in the health care setting" calls for use of alcohol hand rubs, foams, or gels as the primary method to clean hands, except when hands are visibly soiled.i l programs that have been successful in improving hand hygiene and decreasing nosocomial infection have used multidisciplinary teams to develop interventions focusing on use of the alcohol rubs in the settin of institutional commitment and support for the initiativetj ~ hcws should wash hands and forearms to the elbows on arrival in the nursery. a -minute scrub has been suggested?l but consensus on optimal duration of initial hand hygiene is lacking. at a minimum, the initial wash should be long enough to ensure thorough washing and rinsing of all parts of the hands and forearms. routine hand washing throughout care delivery should consist of wetting the hands, applying product, rubbing all surfaces of the hands and fingers vigorously for at least seconds, rinsing, and patting dry with disposable t we s.l~~ wearing hand jewelry has been associated with increased microbial load on hands. whether this results in increased transmission of pathogens is not known. many experts, however, recommend that hand and wrist jewelry not be worn in the in addition, the cdc guideline states that staff who have direct contact with infants in nicus should not wear artificial fingernails or nail extenders."' only natural nails kept less than y.. inch long should be allowed. clean, nonsterile gloves are to be worn whenever contact with blood, body fluids, secretions, excretions, and contaminated items is anticipated. the hcw should change gloves when moving from dirty to clean tasks performed on the same patient, such as after changing a diaper and before suctioning a patient, and whenever they become soiled. because hands can become contaminated during removal of gloves, and because gloves may have tiny, unnoticeable defects, wearing gloves is not a substitute for hand hygiene. hand hygiene must be performed immediately after glove removal. personnel in nurseries including the nicu historically have worn cover gowns for all routine patient contact. the practice has not been found to reduce infection or colonization in neonates and is u n n e c e~s a r y ? '~~~~~ instead, cdc guidelines recommend nonsterile, fluid-resistant gowns to be worn as barrier protection when soiling of clothing is anticipated and in performing procedures likely to result in splashing or spraying of body substance^.^^ possible examples of such procedures in the nicu are placing an arterial line and irrigating a wound. the perinatal guidelines of the american academy of pediatrics and the american college of obstetricians and gynecologists recommend that a longsleeved gown be worn over clothing when a neonate is held outside the bassinette by nursery personnel.'" nonsterile masks, face shields, goggles, and other eye protectors are worn in various combinations to provide barrier protection and should be used during procedures and patient care activities that are likely to generate splashes or sprays of body substances and fl~ids.'~ standard precautions also require that reusable patient care equipment be cleaned and appropriately reprocessed between patients; that soiled linen be handled carefully to prevent contamination of skin, clothing, or the environment; that sharps (i.e., needles, scalpels) be handled carefully to prevent exposure to blood-borne pathogens; and that mouthpieces and other resuscitation devices be used, rather than mouth-to-mouth methods of re~uscitation.'~ in addition to standard precautions, which must be used for every patient, the cdc recommends transmission-based precautions when the patient is known or suspected to be infected or colonized with epidemiologically important or highly transmissible organisms. always used in addition to standard precautions, transmission-based precautions comprise three categories: contact precautions, droplet precautions, and airborne precautions. contact precautions involve the use of barriers to prevent transmission of organisms by direct or indirect contact with the patient or contaminated objects in the patient's immediate e n~i r o n m e n t .~~ sources of indirect contact transmission in nurseries can include patient care equipment such as monitor leads, thermometers, isolettes, breast pumps,le toys, and instruments and contaminated hands. the patient requiring contact precautions should be placed in a private room whenever possible but, after consultation with an infection control practitioner, can be cohorted with a patient infected with the same microorganism but no other infection. many nurseries, however, have few if any isolation rooms. the american academy of pediatrics states that infected neonates requiring contact precautions can be safely cared for without an isolation room if staffing is adequate to allow appropriate hand hygiene, a -to -footwide space can be provided between care stations, adequate hand hygiene facilities are available, and staff members are well trained regarding infection transmission modes.'" hcws should wear clean, nonsterile gloves when entering the room or space of a patient requiring contact precautions and should wear a cover gown when their clothing will have contact with the infant, environmental surfaces, or items in the infant's area. a cover gown also should be worn when the infant has excretions or secretions that are not well contained, such as diarrhea or wound drainage, which may escape the diaper or dressing. infant care equipment should be dedicated to the patient if possible so that it is not shared with thers. ~ examples of conditions in the neonate that require contact precautions include neonatal mucocutaneous herpes simplex virus infection, respiratory syncytial virus infection, varicella (also see airborne precautions), infection or colonization with a resistant organism such as mrsa or a multiple drug-resistant gram-negative bacillus, and congenital rubella syndrome. droplet precautions are intended to reduce the risk of transmission of infectious agents in large-particle droplets from an infected person. such transmission usually occurs when the infected person generates droplets during coughmg, sneezing, or talking, or during procedures such as suctioning. these relatively large droplets travel only short distances and do not remain suspended in the air, but can be deposited on the conjunctiva, nasal mucosa, andfor mouth of persons working within feet of the infected patient. patients requiring droplet precautions should be placed in private rooms (see earlier discussion of isolation rooms in nurseries in the paragraph on contact precautions), and staff should wear masks when working within feet of the patient. examples of conditions in the neonate that necessitate droplet precautions are pertussis and invasive n. meningitidis infection. airborne precautions are designed to reduce the risk of airborne transmission of infectious agents. because of their small size, airborne droplet nuclei and dust particles containing infectious agents or spores can be widely spread on air currents or through ventilation systems and inhaled by or deposited on susceptible hosts. special air-handling systems and ventilation are required to prevent transmission. patients requiring airborne precautions should be placed in private rooms in negative air-pressure ventilation with to air changes per hour. air should be externally exhausted or subjected to high-efficiency particulate air (hepa) filtration if it is recirculated. examples of conditions in the neonate for which airborne precautions are required are varicella-zoster virus infections and measles. susceptible hcws should not enter the rooms of patients with these viral infections. if assignment cannot be avoided, susceptible staff members should wear masks to deliver care. if immunity has been documented, staff members need not wear masks. airborne precautions also are required for active pulmonary tuberculosis, and although neonates are rarely contagious, the cdc recommends isolating patients while they are being e~aluated.~'~ a more important consideration is the need to isolate the family of a suspected tuberculosis patient until an evaluation for pulmonary tuberculosis has been completed, because the source of infection frequently is a member of the child's before the s, well-baby nurseries and many nicus were constructed as large, brightly lit open wards with rows of incubators surrounded by equipment. sinks could be provided in such rooms only around the periphery, limiting access to hand hygiene facilities for staff and families. in these nicus, parents' time with their infant was severely restricted, and the units were designed for the convenience and function of the hcw. more recently, perinatal care professionals have come to understand that neonates (and especially preterm infants) can benefit from a quiet, soothing atmosphere and protection from unnecessary light, noise, handling, uncomfortable positioning, and sleep disruptions? if infants are kept in a central nursery rather than roomingin with mothers, at least square feet of floor space should family. ,z be provided per neonate, and bassinets should be at least feet apart. teams designing units delivering higher levels of perinatal care, including nicus, should plan individual bed areas large enough for families to stay at the bedside for extended periods of time without interfering with the staffs ability to care for the child. if individual rooms cannot be provided, at least square feet of floor space should be allowed for each neonate in an nicu, incubators or overhead warmers should be separated by at least to feet, and aisles should be at least feet a scrub sink with foot, knee, or touchless (electronic sensor) controls should be provided at the entrance to every nursery and should be large and deep enough to control splashing. sinks in patient care areas should be provided at a minimum ratio of sink for at least every to stations in the well-baby nursery and sink for every or stations in higher-level nurseries, including the nicu.'i every bed position should be within feet of a hand-washing sink and accessible for children and persons in wheelchairs. for nicus composed of individual rooms, a hand-washing sink should be located in each room near the door to facilitate hand hygiene on entering and leaving the room. environmental surfaces should be designed so that they are easy to clean and do not harbor microorganisms. sink taps and drains, for instance, have been implicated in outbreaks of infection. z installing sinks with seamless construction may minimize this risk by decreasing areas where water can pool and microorganisms proliferate. faucet aerators have been implicated in outbreaks of infection and should be avoided in the intensive care unit. although carpeting can reduce noise levels in a busy nicu, the cdc "guidelines for environmental infection control in health-care facilities" recommend against use of carpeting in areas where spills are likely, including intensive care units. the guidelines further recommend against upholstered furniture in nicus.~~' if, for reasons of noise reduction and developmentally appropriate care, porous surfaces such as carpeting and cloth upholstery are selected for the nicu, cleaning must be performed carefully. carpet should be vacuumed regularly with equipment fitted with hepa filters, and upholstered furniture should be removed from inpatient areas to be cleaned. attention also should be paid to air-handling systems. according to the perinatal guidelines, minimal standards for inpatient perinatal care areas include six air changes per hour, and a minimum of two changes should consist entirely of outside air. air delivered to the nicu should be filtered with at least % efficiency. in addition, nurseries should include at least one isolation room capable of providing negative pressure vented to the outside, observation windows with blinds for privacy, and the capability for remote m~n i t o r i n g .~~~'~~~ floors and other horizontal surfaces should be cleaned daily by trained personnel using environmental protection agency (epa)-registered hospital disinfectantddetergents. these products (including phenolics and other chemical surface disinfectants) must be prepared in accordance with manufacturers' recommendations and used carefully to avoid exposing neonates to these products. phenolics should not be used on surfaces that come in direct contact with neo-nates' skin. high-touch areas, such as counter tops, work surfaces, doorknobs, and light switches, may need to be cleaned more frequently because they can be heavily contaminated during the process of delivering care. hard, nonporous surfaces should be "wet dusted" rather than dry dusted, to avoid dispersing particulates into the air, and then disinfected using standard hospital disinfectant^.'^^ sinks should be scrubbed daily with a disinfectant detergent. walls, windows, and curtains should be cleaned regularly to prevent dust accumulation, but daily cleaning is not necessary unless they are visibly soiled. bassinets and incubators should be cleaned and disinfected between infants, but care must be taken to rinse cleaning products from surfaces with water before use. care units should not be cleaned with phenolics or other chemical germicides during an infant's stay. instead, infants who remain in the nursery for long periods of time should periodically be moved to freshly cleaned and disinfected units. ' patient care equipment must be cleaned, disinfected, and, when appropriate, sterilized between patients. sterilization (required for devices that enter the vascular system, tissue, or sterile body cavities) and higher levels of disinfection (required for equipment that comes in contact with mucous membranes or that has prolonged or intimate contact with the newborn's skin) must be performed under controlled conditions in the central processing department of the hospital. examples of patient care equipment that require these levels of processing are endotracheal tubes, resuscitation bags, and face masks. , , low-level disinfection is required for less critical equipment, such as stethoscopes or blood pressure cuffs, and usually can be performed at point of use (e.g., the bedside), although this type of equipment should be dedicated to individual patients whenever possible. requirements for linen handling and management for neonates do not vary appreciably from those for other hospitalized patients. although soiled linen can contain large numbers of organisms capable of causing infections, transmission to patients appears to be rare. studies suggesting linen as a source of infection often have failed to confirm it as the source of infection. at least one report, however, has implicated linen in the transmission of group a streptoco~ci.~~ investigation of this outbreak revealed that clothing worn by the neonates was being washed in the local hospital "mini laundry," rather than being processed under the usual laundry contract. investigation of the dryers revealed extensive contamination with the outbreak organism. this case illustrates the importance of having standard hospital laundry protocols and ensuring that appropriate water and dryer temperatures are maintained. when such protocols are followed, the mechanical actions of washing and rinsing, combined with hot water and/or the addition of chemicals such as chlorine bleach, and a final commercial dryer and/or ironing step significantly reduce bacterial few hospitals in the united states use cloth diapers, but regardless of type used, soiled diapers should be carefully bagged in plastic and removed from the unit every hours. hcws caring for neonates have the potential both to transmit infections to infants and to acquire infections from vaccine should be considered for all hcws, including those born before , who have no proof of immunity (receipt of doses of live vaccine on or after first birthday, physician-diagnosed measles, or serologic evidence of immunity). hcws believed t o be susceptible can be vaccinated; adults born before can be considered immune. hcws, both male and female, who lack documentation of receipt of vaccine on or after first birthday or serologic evidence of immunity should be vaccinated; adults born before can be considered immune, except women of childbearing age. hcws without a reliable history of varicella or serologic evidence of varicella immunity should be vaccinated. patients. educating hcws about infection control principles is crucial to preventing such transmission. hospitals should provide education about infection control policies, procedures, and guidelines to staff in all job categories during new employee orientation and on a regular basis throughout employment. the content of this education should include hand hygiene, principles of infection control, the importance of individual responsibility for infection control, and the importance of collaborating with the infection control department in monitoring and investigating potentially harmful infectious exposures and outbreaks. transmission of infectious organisms between patients and hcws has been well documented. several studies have indicated that a high proportion of hcws acquire rsv ( % to %) when working with infected children, and these workers appear to be important in the spread of the illness within hospital^.^^'.^^^ although % of the infected hcws in one of these rsv studies were asymptomatic, staff should be aware of the importance of self-screening for communicable disease. they should be encouraged to report personal infectious illnesses to supervisors, who in turn should report them to occupational health services and infection control. in general, hcws with respiratory, cutaneous, mucocutaneous, or gastrointestinal infections should not deliver direct patient care to neonates. i in addition, seronegative staff members exposed to illnesses, such as varicella and measles, should not work during the contagious portion of the incubation period. staff members with hsv infection rarely have been implicated in transmission of the virus to infants and thus do not need to be routinely excluded from direct patient care. those with herpes labialis or cold sores should be instructed to cover the lesions and not to touch their lesions, and to comply with hand hygiene policies. persons with genital lesions also are unlikely t o transmit hsv so long as hand hygiene policies are followed. however, hcws who are unlikely or unable to comply with the infection control measures and those with herpetic whitlow should not deliver direct patient care to neonates until lesions have healed. acquisition of cmv often is a concern of pregnant hcws because of the potential effect on the fetus. approximately % of newborn infants in most nurseries and a higher percentage of older children (up to % of children to years of age in child-care centers) excrete cmv without clinical manifestation^.'^^ the risk of acquiring cmv infection has not been shown to be higher for hcws than for the general for this reason, pregnant caregivers need not be excluded from the care of neonates suspected to be shedding cmv. they should be advised of the importance of standard precautions. hcws in well-baby nurseries and nicus should be as free from transmissible infectious diseases as possible? and ensuring that they are immune to vaccine-preventable diseases is an essential part of a personnel health program. the cdc recommends several immunizations for health care personnel (table - ) . staffing levels in a patient care setting also can affect patient outcomes. a number of studies suggest that as patient-tonurse ratios in intensive care units increase, so do nosocomial infections and mortality rate^.^^,'^^,'^ although optimal staffing ratios have not been established for nicus and will vary according to characteristics of individual units, one study demonstrated that the incidence of clustered s. aureus infections was times higher after periods when the infantto-nurse ratio exceeded : . decreased compliance with hand hygiene during a period of understaffing frequently is cited as contributing to nosocomial infection rate increases." further study is necessary to determine best practice surrounding staffing levels in nicus. the first nicus in the late s grouped infants together in large, brightly lit rooms with incubators placed in rows. parents were allowed very little time with their babies and even less physical contact. in the decades since, it has been recognized that "the parent is the most important caregiver and constant influence in an infant's life" and that hcws working in nicus should encourage parents to become involved in the nonmedical aspects of their child's care. principles of family-centered care also include liberal nicu visitation for relatives, siblings, and family friends and the involvement of parents in the development of nursery policies and programs that promote parenting skills. care must be taken, however, to minimize risk of infection for the neonate. mothers can transmit infections to neonates both during delivery and post partum, although separation of mother and newborn rarely is indicated. in the absence of certain specific infections, mothers, including those with postpartum fever not attributed to a specific infection, should be allowed to handle their infants if the following conditions are met: they feel well enough. they wash their hands well under supervision. a clean gown is worn. contact of the neonate with contaminated dressings, a mother with a transmissible illness not requiring separation from her infant should be carefully educated about the mode of transmission and precautions necessary to protect her infant. personal protective equipment, such as cover gowns, gloves, and masks, and hand hygiene facilities should be readily available to her, and she should perform hand hygiene and don a long-sleeved cover gown before handling her infant. if wounds or abscesses are present, drainage should be contained within a dressing. if drainage cannot be completely contained, separation from the infant may be necessary. care should be taken to prevent the infant from coming in contact with soiled linens, clothing, dressings, or other potentially contaminated items. the mother with active genital hsv lesions need not be separated from her infant if the foregoing precautions are taken. those with herpes labialis should not kiss or nuzzle their infants until lesions have cleared; lesions should be covered and a surgical mask may be worn until the lesions are crusted and dry, and careful hand hygiene should be stressed. mothers with viral respiratory infections should be made aware that many of these illnesses are transmitted by contact with infected secretions as well as by droplet spread, that soiled tissues should be disposed of carefully, and that hand hygiene is critical to transmission prevention. masks can be worn to reduce the risk of droplet t r a n s m i s s i~n .~~~'~~~ as previously mentioned, although very few infections require separation of mother and infant, women with untreated active pulmonary tuberculosis should be separated from their infants until they no longer are contagious. mothers with group a streptococcal infections, especially when involving draining wounds, also should be isolated from their infants until they no longer are contagious. less certain is the necessity of separating mothers with peripartum varicella (onset of infection within days before or days after delivery) from their uninfected infants. the perinatal guidelines recommend that such infants remain with their mothers after receiving varicella-zoster immune globulin (vzig) but caution that infant and mother must be carefully managed in airborne and contact precautions to prevent transmission within the nursery. some experts recommend separating these mothers from their infants until all lesions are dried and crusted. numerous studies support the value of human milk for infants (see chapter ). besides providing optimal nutritional content for infants, it has been shown to be associated with a lower incidence of infections and sepsis in the first year of linen, clothing, or pads is avoided. although contraindications to breast-feeding are few, mothers who have active untreated tuberculosis, human immunodeficiency virus (hiv) infection, breast abscesses (as opposed to simple mastitis that is being treated with antimicrobial therapy), or hsv lesions around the nipples should not breast-feed. mothers who are hepatitis b surface antigen positive may breast-feed, because ingestion of an infected mother's milk has not been shown to increase the risk of transmission to her child, but the infant must receive hepatitis b virus immune globulin (hbig) and vaccine immediately after birth. because systemic disease may develop in preterm infants with low concentrations of transplacentally acquired antibodies to cmv following ingestion of milk of cmv-seropositive mothers, decisions regarding breast-feeding should consider the benefits of human milk versus the risk of cmv transmission. freezing breast milk has been shown to decrease viral titers but does not eliminate cmv; pasteurization of human milk can inactivate cmv. either method may be considered in attempts to decrease risk of transmission for breast-feeding nicu neonates. neonates in the nicu frequently are incapable of breastfeeding because of maternal separation, unstable respiratory status, and immaturity of the sucking reflex. for these reasons, mothers of such infants must use a breast pump to couect milk for administration through a feeding tube. pumping, collection, and storage of breast milk create opportunities for contamination of the milk, and for cross-infection if equipment is shared between mothers. several studies have demonstrated contamination of breast pumps, contamination of expressed milk that had been frozen and thawed, and higher levels of stool colonization with aerobic bacteria in infants fed precollected breast milk. , * consensus is lacking on the safe level of microbiologic contamination of breast milk, and most expressed breast milk contains normal skin flora. although breast milk containing greater than cfu/ml of gram-negative bacteria has been reported to cause feeding intolerance and to be associated with suspected sepsis, routine bacterial culturing of expressed breast milk is not recommended. v instead, efforts to ensure safety of expressed milk should focus on optimal collection, storage, and administration techniques. cleaning and disinfection of breast pumps should be included in educational material provided to nursing mothers (table - ). in addition, mothers should be instructed to perform hand hygiene and cleanse nipples with cotton and plain water before expressing milk in sterile containers. expressed breast milk can be refrigerated for up to hours and can be safely frozen (- " c f °c [- " f f . " f]) for up to months. it can be thawed quickly under warm running water (avoiding contamination with tap water) or gradually in a refrigerator. exposure to high temperatures, as may be experienced in a microwave, can destroy valuable components of the milk. thawed breast milk can be stored in the refrigerator for up to hours before it must be discarded. to avoid proliferation of microorganisms, milk administered through a feeding tube by continuous infusion should hang no longer than to hours before replacement of the milk, container, and for mothers who choose not to breast-feed, commercial infant formula is available. most hospitals now use sterile, ready-to-feed formulas provided by the manufacturer in bottles, with sterile nipples to attach just before use. nipples each mother is supplied with a personal pumping kit. nursing staff instruct mothers in techniques of milk expression and appropriate procedures for cleaning breast pump parts: wipe all horizontal surfaces at the pumping station with hospital disinfectant before and after pumping. wash hands with soapy water before and after pumping. wash all parts of the breast pump kit that have been in contact with milk in hot water and dish detergent or in a dishwasher. expressed milk is collected in sterile, single-use plastic (polycarbonate or polypropylene) containers. breast milk containers are labeled with infant's name and the date and time of collection. administration containers (bottle or syringe) are similarly labeled when breast milk is transferred from collection containers. all hcws wear gloves when handling and administering breast milk. two persons check t h e labeled administration container against the infant's hospital identification band before administering breast milk (may be two hcws or one hcw and a family member). hcw. health care worker. from infection control policy, children's hospital and regional medical center, seattle, are best attached at the bedside just before feeding, and the unit should be used immediately and discarded within hours after the bottle is ~ncapped.'~' specialty and less commonly used formulas may not be available as a ready-to-feed product, and breast milk supplements do not come in liquid form. after a recent report of a case of fatal enterobacter sakazakii meningitis in a neonate fed contaminated powdered infant formula: concerns have risen about the safety of these products. although powdered formulas are not sterile, preparation and storage practices can decrease the possibility of proliferation of microorganisms after preparation. the cdc, the food and drug administration, and the american dietetic association offered updated recommendations on the safe preparation and administration of commercial formula after the recall of the product linked to the e. sakazakii case. these recommendations instruct the care provider as follows: use alternatives (ready-to-feed or concentrated formulas) to powdered infant formula whenever possible. prepare formula using aseptic technique in a designated formula preparation room. refrigerate prepared formula so that a temperature of " to " c is reached by hours after preparation, and discard any reconstituted formula stored longer than hours. limit ambient-temperature hang time of continuously infused formula to no longer than hours. use hygienic handling techniques at feeding time, and avoid open delivery systems. have written guidelines for managing a manufacturer's recall of contaminated formula. the fda also recommended that boiling water be used to prepare powdered formulas, but concerns about this recommendation include potential damage to formula components from the high temperature of the water, a lack of evidence that using this method would lull potential pathogens in the formula, and risk of injury to persons preparing the f rmula. ~' the concept of co-bedding, or the bunlung of twin infants (or other multiples) in a single isolette or crib, is being explored in nicus for the potential benefits offered to the babies. co-bedding as a component of developmentally supportive care is based on the premise that extrauterine adaptation of twin neonates is enhanced by continued physical contact with the other twin. potential benefits need further study but may include increased bonding, decreased need for temperature support, and easier transition to home. it is certainly possible, however, for one of a set of multiples to be infected while the others are not, and for parents to be implicated as vectors in infection transmission. it also is possible for invasive devices and intravascular catheters to be dislodged by close contact with an active sibling. therefore, exclusion criteria for co-bedding infants should include clinical findings suggesting infection that could be transmitted to a sibling (e.g., draining wound) and the need for drains and central venous or arterial line^.*^^-'^^ the principles of family-centered care encourage liberal visitation policies, both in the well-baby nursery (or roomingin scenario) and in the nicu. parents, including fathers, should be allowed unlimited visitation to their newborns, and siblings also should be allowed liberal visitation. expanding the number of visitors to neonates may, however, increase the risk of disease exposure if education and screening for symptoms of infection are not implemented. written policies should be in place to guide sibling visits, and parents should be encouraged to share the responsibility of protecting their newborn from contagious illnesses. the perinatal guidelines regarding persons who visit newborns are listed in table - . adult visitors to neonates, including parents, have been implicated in outbreaks of infections including p aeruginosa infection, pertussis, and salmonella i n f e c t i~n . '~'~~~~~~~~ ac cordingly, the principles for sibling visitation should be applied to adult visitors as well. they should be screened for symptoms of contagious illness, instructed to perform hand hygiene before entering the nicu and before and after touching the neonate, and should interact only with the family member they came to the hospital to visit. families of neonates who have lengthy nicu stays may come to know each other well and serve as sources of emotional support to one another. nevertheless, they should be educated about the potential of transmitting microorganisms and infections between families if standard precautions and physical separation are not maintained, even though they may be sharing an inpatient space. sibling visits should be encouraged for healthy and ill newborns. parents should be interviewed at a site outside the nursery to establish that the siblings are not ill before allowing them to visit. children with fever or other symptoms of an acute illness such as upper respiratory infection or gastroenteritis, or those recently visiting children should visit only their sibling. children should be prepared in advance for their visit. visitors should be adequately observed and monitored by hospital staff. children should carefully wash their hands before patient contact. throughout t h e visit, siblings should be supervised by parents or another responsible adult. exposed to a known communicable disease such as chickenpox, should not be allowed to visit. bathing the newborn is standard practice in nurseries, but very little standardization in frequency or cleansing product exists. if not performed carefully, bathing actually can be detrimental to the infant, resulting in hypothermia, increased crying with resulting increases in oxygen consumption, respiratory distress, and instability of vital signs. although the initial bath or cleansing should be delayed until the neonate's temperature has been stable for several hours, removing blood and drying the skin immediately after delivery may remove potentially infectious microorganisms such as hepatitis b virus, hsv, and hiv, minimizing risk to the neonate from maternal infection. when the newborn requires an intramuscular injection in the delivery room, infection sites should be cleansed with alcohol to prevent transmission of organisms that may be present in maternal blood and body for routine bathing in the first few weeks of life, plain warm water should be used. this is especially important for preterm infants, as well as full-term infants with barrier compromise such as abrasions or dermatitis. if a soap is necessary for heavily soiled areas, a mild ph-neutral product without additives should be used, and duration of soaping should be restricted to less than minutes no more than three times per week. few randomized studies comparing cord care regimens and infection rates have been performed, and consensus has not been reached on best practice regarding care of the umbilical cord stump. a review published in described care regimens used for more than decades, including combinations of triple dye, chlorhexidine, % alcohol, bacitracin, hexachlorophene, povidone-iodine, and "dry care" (soap and water cleansing of soiled periumbilical skin) and found variable impact on colonization of the stump. the study authors suggested that dry cord care alone may be insufficient and that chlorhexidine seemed to be a favorable antiseptic choice for cord care because of its activity against gram-positive and gram-negative bacteria. they went on to stress, however, that large, well-designed studies were required before firm conclusions could be drawn. the current perinatal guidelines do not recommend a specific regimen but warn that use of alcohol alone is not an effective method of preventing umbilical cord colonization and ~mphalitis.'~~ the perinatal guidelines further recommend that diapers be folded away from and below the stump and that emollients not be applied to the although blindness resulting from neonatal conjunctivitis is rare in the united states, with a reported incidence of . % or less, the rate among the million infants born annually throughout the world is as high as %. chlamydia trachornatis has been the most common etiologic agent in the united states, but other organisms such as neisseria gonorrhoeae, s. aureus, and e. coli also can cause ophthalmia neonatorum. use of % silver nitrate drops, at one time the agent of choice, is no longer recommended because of concerns about associated chemical irritation. agents thought to be equally efficacious and now recommended include % tetracycline and . % erythromycin ophthalmic ointments, administered from sterile single-use tubes or vials. ~ povidone-iodine ( . %) ophthalmic solution also can be used and in one study was shown to be more effective than silver nitrate or erythromycin in the prevention of ophthalmia neonatorum. bacterial resistance has not been seen with this agent, it causes less toxicity than either silver nitrate or erythromycin, and it is less expensivea definite consideration in developing countries. whatever the agent selected, it should reach all parts of the conjunctival sac, and the eyes should not be irrigated after application. ophthalmic agents will not necessarily prevent ocular or disseminated gonorrhea in infants born to mothers with active infection at time of delivery. these infants should be given parenteral antimicrobial therapy as well as ocular p r o p h y l a~i s . '~~,~~~ some experts also advise giving infants born to mothers with untreated genital chlamydia infections a course of oral erythromycin beginning on the second or third day of life. primary bloodstream infections (defined by the cdc nnis system as being due to a pathogen cultured from one or more blood specimens not related to an infection at another site) account for a large proportion of infections in nicu infants:' and most are related to the use of an intravascular catheter. peripheral intravenous catheters (pivs) are the most frequently used devices for the neonate for intravenous therapy of short duration. when longer access is necessary, nontunneled cvcs such as umbilical catheters and piccs most commonly are the most recent data available conduct surveillance in nlcus to determine catheter-related bloodstream infection rates, monitor trends, and identify infection control lapses. investigate events leading to unexpected life-threatening or fatal outcomes. select the catheter, insertion technique, and insertion site with the lowest risk for complications for the anticipated type and duration of intravenous therapy. use a cvc with the minimal number of ports essential f o r management of t h e patient. designate one port for hyperalimentation if a multilumen catheter is used. educate hcws who insert and maintain catheters, and assess their knowledge and competence periodically. use aseptic technique and maximal sterile barriers during insertion of cvcs (cap, mask, sterile gown, sterile gloves, and a large sterile barrier). do not routinely replace cvcs, piccs, or pulmonary artery catheters to prevent catheter-related infections. do not remove on the basis of fever alone. in pediatric patients, leave peripheral venous catheters in place until intravenous therapy is completed unless a complication (e.g., phlebitis, infiltration) occurs. remove intravascular catheters promptly when no longer essential. observe proper hand hygiene procedures either by washing with antiseptic-containing soap and water or use of waterless alcohol-based products before and after working with intravascular lines. disinfect skin with an appropriate antiseptic before catheter insertion and during dressing changes. a % chlorhexidine-based preparation is preferred. do not use topical antibiotic ointment or creams on insertion sites, except when using dialysis catheters. use either sterile gauze or sterile, transparent, semipermeable dressing to cover t h e catheter site. replace gauze dressings on short-term cvc sites every days and transparent dressings at least weekly, except in pediatric patients, in whom the risk of dislodging the catheter outweighs the benefit of changing the dressing. change if damp, loosened, or visibly soiled. replace dressings on tunneled or implanted cvc sites no more than once per week until the insertion site has healed. chlorhexidine sponge dressings are contraindicated in neonates younger than days or those born at a gestational age of less than weeks. clean injection ports with % alcohol or an iodophor before accessing the system. use disposable transducer assemblies with peripheral arterial catheters and pressure monitoring devices. keep all components of such systems sterile, and do not administer dextrose-containing solutions or parenteral nutrition fluids through them. from nnis (august ) revealed that the mean umbilical catheter-and cvc-associated bloodstream infection rates for nicus ranged from . per catheter days for infants whose birth weight was less than g to . per catheter days in infants whose birth weight was g or more.' the cdc recommends implementing strategies to reduce the incidence of such infections that strike a balance between patient safety and cost-effectiveness. few large studies of risks related to intravascular devices have been performed in nicu patients. as a result, intravascular device recommendations for neonates are based on those developed for adults and older pediatric patients (table - ) . several differences in their management should be considered. although the cdc recommends, in certain circumstances, using antimicrobialor antiseptic-impregnated cvcs in adults whose catheters are expected to remain in place more than days, these catheters are not available in sizes small enough for neonates. of more importance, studies to evaluate their safety in neonates, especially premature neonates of very low birth weight, have not been performed. in addition, although the cdc recommends changing the insertion site of pivs at least every to hours in adults, data suggest that leaving pivs in place in pediatric patients does not increase the risk of complications. the cdc guidelines recommend that pivs be left in place in children until therapy is completed, unless complications occur. careful skin antisepsis before insertion of an intravascular catheter is critical to prevention of intravascular devicerelated bacteremia, although care in the selection of a product for use on neonatal skin is required. chlorhexidine preparations are recommended by the cdc because these products have been found to be superior to povidone-iodine in reducing the risk for peripheral catheter colonization in neonates. residues left on the skin by chlorhexidine prolong its half-life, providing improved protection for catheters in neonates that must be left in place for longer periods of timef ' umbilical veins and arteries are available for cvc insertion only in neonates and are typically used for several days; thereafter, the cvc is replaced with another, nontunneled cvc or picc if continued central venous access is required. the umbilicus provides a site that can be cannulated easily, allowing for collection of blood specimens and hernodynamic measurements, but after birth, the umbilicus quickly becomes heavily colonized with skin flora and other microorganisms. colonization and catheter-related bloodstream infection rates for umbilical vein and umbilical artery catheters are similar. colonization rates for umbilical artery catheters are estimated to be % to %; the estimated rate for umbilical artery catheter-related bloodstream infection is %? colonization rates are from % to % for umbilical vein catheters; rates for umbilical vein catheter-related bloodstream infections are % to ? .~~ a summary of the cdc recommendations for management of umbilical catheters is presented in table - . as mentioned earlier, nnis data indicate that nosocomial pneumonia is the second most common infection type in cleanse umbilical insertion site with an antiseptic before catheter insertion. avoid tincture of iodine; povidone-iodine can be used. add low doses of heparin to fluid infused through umbilical artery catheters. remove and do not replace umbilical catheters if signs of catheter-related bloodstream infection, vascular insufficiency, or remove umbilical catheters as soon as possible when no longer needed or if any sign of vascular insufficiency to the lower umbilical artery catheters should not be left in place for longer than days. umbilical venous catheters should be removed as soon as possible when no longer needed but can be used for up to nicu patients. risk factors for ventilator-associated pneumonia can be grouped as host-related (prematurity, low birth weight, sedation or use of paralytic agents), devicerelated (endotracheal intubation, mechanical ventilation, orogastric or nasogastric tube placement) and factors that increase bacterial colonization of the stomach or nasopharynx (broad-spectrum antimicrobial agents, antacids, or h, b l o c k e r~) . ~,~"~~~ ventilator-associated pneumonia generally refers to bacterial pneumonia that develops in patients who are receiving mechanical ventilation. aspiration and direct inoculation of bacteria are the primary routes of entry into the lower respiratory tract; the source of these organisms may be the patient's endogenous flora or transmission from other patients, staff members, or the e n~i r o n m e n t .~~'~~~ few studies have been performed to assess the effectiveness of prevention strategies in pediatric patients. strategies to prevent ventilator-associated pneumonia in the nicu patient are therefore based primarily on studies performed in adults (table - ) . hand hygiene remains critical to the prevention of ventilator-associated pneumonia, and hcws should consistently apply the principles of standard precautions to the care of the ventilated patient, wearing gloves to handle respiratory secretions or objects contaminated by them, and changing gloves and performing hand hygiene between contacts with a contaminated body site and the respiratory tract or a respiratory tract device. because mechanical ventilation is a significant risk factor for the development of nosocomial infection or ventilatorassociated pneumonia, weaning from ventilation and removing endotracheal tubes as soon as indication for their use ceases are key infection control strategies. as an alternative to endotracheal intubation, noninvasive nasal continuous positive airway pressure (cpap) ventilation avoids some of the common risk factors for ventilator-associated pneumonia and has been used successfully for neonate^?^^"^^ respiratory care equipment that comes in contact with mucous membranes of ventilated patients or that is part of the ventilator circuit should be single use (discarded after one-time use with a single patient) or be subjected to sterilization or high-level disinfection between patients. wet heat pasteurization (processing at oc for minutes) or chemical disinfectants can be used to achieve high-level disinfection of reusable respiratory equipment. ventilator circuits should be changed no more frequently than every hours, and evidence suggests that extending the length of time between changes to days does not increase the risk of ventilator-associated pneumonia.z s circuits should be monitored for accumulation of condensate and drained periodically, with care taken to avoid allowing the condensate, a potential reservoir for pathogens, to drain toward the sterile fluids should be used for nebulization, and sterile water should be used to rinse reusable semicritical equipment and devices such as in-line medication nebulizers. basic infection control measures, such as hand hygiene and wearing gloves during suctioning and respiratory manipulation, also can reduce the risk of nosocomial pneumonia. both open, single-use and closed, multiuse suction systems are available. if an open system is used, a sterile single-use catheter should be used each time the patient is suctioned. closed systems, which do not need to be changed daily and can be used for up to have the advantage of lower costs and decreased environmental cross-contamination but have not been shown to decrease the incidence of nosocomial pneumonia when compared with open systems. v although not well studied in pediatric patients, aspiration of oropharyngeal secretions is believed to contribute to development of ventilator-associated pneumonia in adults. placing the mechanically ventilated patient in a semirecumbent position or elevating the head of the bed in an attempt to minimize aspiration is recommended unless medically contraindicated. also, placement of enteral feeding tubes should be verified before their to prevent regurgitation and potential aspiration of stomach contents by the sedated patient, overdistention of the stomach should be avoided by regular monitoring of the patient's intestinal motility, serial measurement of residual gastric volume or abdominal girth, reducing the use of narcotics and anticholinergic agents, and adjusting the rate and volume of enteral fee ding^.^^^,^^^ oral decontamination, with the intent of decreasing oropharyngeal colonization, has been studied in adults and seems to lower the incidence of ventilatorassociated pneumonia (although not duration of ventilation or mortality but further work is needed to determine whether this is an effective strategy in neonates. in addition, medications such as sucralfate, as opposed to histamine h, receptor antagonists and antacids, which raise gastric ph and can potentially result in increased bacterial colonization of the stomach, have been used to prevent development of stress ulcers and have been associated with lower incidence of ventilator-associated pneumonia in adults. two studies suggest, however, that this approach is of no benefit in pediatric patients, but the authors stress that additional studies with larger sample sizes are needed to confirm these nosocomial infections in a neonatal intensive care unit: incidence and risk factors. am infect control bektas s, goetze b, speer cp. decreased adherence, chemotaxis and phagocytic activities of neutrophils from preterm neonates surgery, sepsis, and nonspecific immune function in neonates. pediatr surg - , . stiehm er. the physiologic immunodeficiency of immaturity diminished interferongamma and lymphocyte 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liu, aihan; jiang, hezhishi; guo, jing; yuan, xiumei; zhang, yun; liu, liu; liu, yu title: cost-effectiveness analysis of antiepidemic policies and global situation assessment of covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: qp o g with a two-layer contact-dispersion model and data in china, we analyze the cost-effectiveness of three types of antiepidemic measures for covid- : regular epidemiological control, local social interaction control, and inter-city travel restriction. we find that: ) intercity travel restriction has minimal or even negative effect compared to the other two at the national level; ) the time of reaching turning point is independent of the current number of cases, and only related to the enforcement stringency of epidemiological control and social interaction control measures; ) strong enforcement at the early stage is the only opportunity to maximize both antiepidemic effectiveness and cost-effectiveness; ) mediocre stringency of social interaction measures is the worst choice. subsequently, we cluster countries/regions into four groups based on their control measures and provide situation assessment and policy suggestions for each group. coronavirus disease (covid - ) has been recognized as a pandemic by the world health organization in march ( ) . nonetheless, on march , mainland china observed their first day with zero increase of local cases since the outbreak ( ) . this indicates that, aside from the imported cases, the pandemic has been locally close to the end. many experiences and lessons can be shared by the rest of the world from the trajectories of the outbreak and the control strategies in mainland china. one question that is of particular importance is the costeffectiveness of different antiepidemic measures. drawn from the chinese experiences and lessons, three categories of measures have been implemented: a) "regular" epidemiological control and prevention measures, including identification of infected cases, tracing their close contacts, and quarantines for both; b) in-city activity restrictions, including work-from-home, shutdown of schools and public spaces, cancellation of events, and lock-down of residential neighborhoods; c) inter-city travel restrictions, including temperature screening at all transportation terminals, cancellation of flights and trains, and eventually travel bans from/to certain cities. specifically, (b) and (c) are considered "irregular," which contain the spread of disease in an aggressive manner through the suppression of all possible social interactions. however, these measures lead to enormous economic loss, which may mean higher unemployment rates, and shortage of food, medical services, and other necessities. those chain effects may also endanger the lives of certain social groups. fundamentally this is a trolley dilemma, and the life and health of human beings can hardly be evaluated using monetary values. nevertheless, a comprehensive understanding of the cost of each measure-including the opportunity cost of the shutdown economic activities-and the effectiveness of lifesaving can still help policy makers to compare different antiepidemic strategies in a more operable way. further, with these insights, we can then perform a cross-sectional assessment of the situation of the global antiepidemic campaign regarding the aforementioned measures in different countries/regions, such that typical policies can be clustered and prescriptive policy suggestions can be provided accordingly, which also echoes the argument in ( ) . in prior research, scholars have used deductive models to find that the timing of lockdown, including both inter-city travel restrictions and social distancing, significantly changes the size of infected population and the spatial extent of spread ( ) ( ) ( ) ( ) . nonetheless, all prior research only focused on specific instances of policies without discussing generalizable impacts of different measures and lacked a cost-effectiveness assessment of each measure ( ) ( ) ( ) . in this article, based on the transmission pattern of covid- in china, we build a two-layer contactspread model ( fig. ) to recover the whole spatio-temporal transmission process, especially the early-stage numbers and distribution of cases at the prefecture level (see sm , , and ). with this model as a generalizable baseline, we conducted a comprehensive sensitivity analysis for all antiepidemic measures (fig. ) and concluded the following assessment on the effectiveness of the measures, in terms of number of infected cases, and number of infected cities. . containing daily social interaction, parameterized in the model as ! , " for the infected and the exposed populations, is the most effective measure for controlling both the number of infected cases and the spatial extent of the spread. more specifically, we find that a) the controls of ! and " show comparable and substitutable effects in containing the spread of disease, with different elasticity in different stages of the epidemic. controlling " is more effective when the number of cases is small (e.g., fewer than in a city), and ! is more effective when the number of cases is sufficiently large. the implication is that at the early stage of transmission, comprehensive epidemic surveys and contact tracing, alongside with strict quarantine and social distancing, should be used to prioritize the reduction of the social interaction levels of the exposed population ( " ), while after the number of cases has increased to a sufficiently high level, comprehensive testing and identification of all infected cases should be prioritized in order to reduce the social interaction levels of the infected population ( ! ). b) stricter control of social interaction for both ! and " have diminishing returns in reducing the number of cases, but increasing returns in limiting the spatial extent of spread. for example, in one month of simulation, when daily social interaction drops from % to % of the normal level, the number of infected cases drops by %- % (depending on the stage of epidemic), while the number of cities with infected cases drops only by - %; when daily social interaction drops from % to , however, the number of cases drops only by . %- . % for infected cases versus %- % for cities with infected cases. an exception is at the ending stage of the epidemic, when controlling social interaction has increasing returns in both effects. c) the effects of the epidemiological and social interaction control measures are monotonic for the reduction of infected cases and the spatial extent of spread: the stricter they are enforced, the lower number of infected cases and the narrower spatial extent of spread can be observed. . the time of reaching turning point is independent of the current number of infected cases but is only related to the stringency of epidemiological and social interaction control measures, i.e., the relative change of ! , " . when the two parameters are / - / of the normal everyday values, the turning point comes in two weeks and the clearance of cases happens in two to three months; when ! , " are larger than / - / of the normal values, the turning point will never come, i.e., the peak value of case numbers will remain the same as if there are no such measures, but they only delay the time of peak. . except at the early stage and the ending stage, inter-city travel restriction has only minimal effect on both the reduction of infected cases and control of disease spread in a city network. overall, compared with in-city epidemiological and social interaction control measures, the contribution of inter-city travel restrictions to the reduction of the number of infected cases and the spatial spread of disease is much smaller-lower by two orders of magnitudes. when the number of cases is sufficiently large, inter-city travel restriction even exacerbates the situation since it limits the social interaction of infected cases, and "condenses" # locally ( ) . this finding is consistent with that in prior research ( , ) . therefore, to national or regional governments who manage a city network, and to international antiepidemic collaboration, travel restriction should only be regarded as an auxiliary measure at the beginning and ending stage of the spread to protect cities which have not been infected at all, or only with a sufficiently small number of cases. in the latter case epidemiological and social interaction control measures should also be implemented simultaneously to get the health care system and other prevention measures prepared. the simulation-based formal analysis above is consistent with the empirical evidence in china. the lockdown of wuhan, and nationwide strict enforcement of epidemiological control and social distancing policies around january , including the cancellation of all chinese new near gatherings mark the key move of the antiepidemic campaign. at that time point, all other cities in china were at early stages of the epidemic, which guaranteed the effectiveness of the wuhan travel ban. in addition, with aggressive social interaction control in all cities the turning point of the number of cases arrived in two weeks outside of wuhan. in wuhan, the key move was the functioning of fāngcāng hospitals (mobile cabin hospitals) in early february which enabled citywide comprehensive quarantine of the infected population ( ) . this measure reduced the social interaction of infected cases to almost zero, and together with strict social distancing they effectively reversed the trend of spread after two weeks. in terms of inter-city travel restrictions, since they were during the chinese new year and the extended holidays, and overlapped with social distancing measures, the net effect could not be easily isolated empirically. nonetheless, since mid-february the economy had re-opened. by the end of march, the inter-city migration in southern and eastern chinese cities had recovered to the same level as in previous years ( ) , but most cities still observed almost zero increase of infected cases. this further supports that the effectiveness of travel restriction is very limited for the cities with small numbers of cases. another evidence to support this point is the , people who left wuhan right before the lockdown night ( ) . this "escaped" population did not significantly change the effectiveness of the national antiepidemic effort. further analysis on the cost-effectiveness of the measures shows more irregularity and non-linearity, leading to more nuanced relationships (detailed in sm ). here we summarize the most critical general patterns as follows: . the measures which can achieve both high antiepidemic effectiveness (low number of cases and narrow spatial spread) and high cost-effectiveness (smaller loss of economic outputs) only exist at the early stage of transmission. at the early stage, if epidemiological and social interaction control measures can be strictly enforced (sufficiently low ! and " ), it is possible to keep the spread at a low level, with a loss of economic outputs only up to %. the intuition is as follows: based on the assumptions of this article, the early-stage measures only include comprehensive testing, close contact tracing, and quarantine, but do not include indiscriminate restrictions of in-city social interaction and inter-city travel, which incurs high costs. the policy implication is straightforward: for early-stage cities and regions, it is critical to practice epidemiological control interventions, but not to necessarily mobilize the whole society into social interaction reduction. this finding is consistent with the suggestions in ( ). . except for the early stage, it is impossible to simultaneously achieve both high antiepidemic effectiveness and high cost-effectiveness. except for a few "plateaus," the effectiveness of epidemiological and social interaction control measures monotonically increases with the stringency of control measures. however, the cost and cost-effectiveness functions are non-monotonic and there usually exists more than one peak (see details in sm ), which in most cases do not coincide with the effectiveness peak. typically, the costs are the lowest when the control measures are at sufficiently low or sufficiently high levels. while the latter case has been explained in the last point, the sufficiently low control measure scenario basically leaves the whole population to be infected. therefore, the tradeoff between sufficiently low and sufficiently high levels of control measures depend on many technological factors, including the short-term and long-term capacity of healthcare systems, long-term uncertainty of virus mutation, and development of vaccines, as well as many non-technological factors, including the risk averse attitudes for the short term and the long term, the mental discounting between short-term and long-term tradeoffs, and the fundamental value judgement on the "value of lives," the discussion of which are beyond the scope of this article, and will be left for discussion at the end of this article. . lastly, although it is difficult to choose the optimal control strategy, the worst choice is explicit: mediocre control of social interaction, e.g., social distancing with leakage. this choice still incurs - % loss of economic outputs, but only achieves - % reduction in the number of cases, an extent which is insufficient to overturn the epidemic curve. except for moderately delaying the spread of disease which may be taken advantage of to get the healthcare system prepared, this strategy is the worst choice in all other dimensions. with the formal results above, we can now perform a cross-sectional assessment of the global situation of the antiepidemic campaign from a transmission-prevention policy perspective. among the three types of measures (epidemiological control measures, social distancing, and travel restriction), we disregard the travel restriction measure as our results clearly show that it is ineffective for most countries/regions under the current situation (we will discuss the exceptions later). rather, we use two datasets ( , ) which codified the antiepidemic measures chosen by countries/regions as of april (due to the lack of testing data, we use wuhan as a proxy for mainland china), and for the countries/regions analyze the relationship of their two stringency indices, $ and % , i.e., the activity levels of the infected and exposed populations, and the respective effectiveness on the reduction of infected cases. based on the stringency of the two dimensions of antiepidemic measures, we can divide all countries/regions into three groups (fig. ) , each with a different antiepidemic "strategy": elimination, control, and delay. more than countries/regions are not included because of the lack of data. we will also discuss the implications of this fact. . the "elimination" group: this group (up right corner of fig. ) consists of only a few countries/regions, including mainland china (represented by wuhan), hong kong sar, vietnam, uae, bahrain, etc., all with # ≪ , such that the epidemic could be expected to dwarf within a reasonably short time period. mainland china is the most prominent example of this group, where aggressive measures have been taken on both dimensions to reduce the activity of the infected population as well as the exposed population. the measures include effective epidemiological control interventions, such as comprehensive testing and close contact tracing, and also aggressive social distancing measures, such as shutdown of schools, workplaces, and public transport, cancellation of events, and mass disease control education. these measures incur %- % loss of economic outcome in a month, and the loss accumulates as the epidemic is not completely "eliminated". obviously, the underlying value judgment of the elimination strategy is an overwhelmingly high weight on health and lives over any cost-control or costeffectiveness reckoning. although the treasuring for lives is always respectable, long-lasting economic tightening also constitutes a threat to society, especially to the disadvantaged social groups. due to the existence of asymptomatic carriers, false-negative test results, and international imports of cases, a complete elimination of the epidemic is extremely difficult. thus, if the aim is to literally eliminate all cases, the economic losses are highly likely to accumulate to an unbearable level. therefore, we suggest that countries/regions which have followed the elimination strategy consider turning to the "control" strategy (elaborated below) to avoid excess economic losses on the condition that the active number of infected cases has been reduced to a sufficiently low level. we also suggest that these countries/regions keep the travel restriction measures-the most effective measure at this stage of the epidemic indicated by our simulation results. . the "control" group: this group includes south korea, singapore, qatar, norway, slovenia, russia, and new zealand, etc., all with # < , but still not sufficiently small, such that the epidemic can be reduced to a lower level (but not eliminated), depending on the stringency of intervention measures. the singapore in february was the most prominent example within this group, where antiepidemic measures have been mild enough not to affect everyday life by aggressive social distancing. through regular epidemiological control practices, they were managed to maintain a daily increase of infected cases fewer than , and only suffered . %- % loss of economic outcome in a month. the control strategy requires a highly capable epidemic control system. given the aforementioned long-term uncertainties, even with such a capable system, the strategy is still a tightrope-walking game with the risk of abrupt system overload by accidentally untracked surges of infection, which, unfortunately, appears to be the case in singapore in early april. under such circumstances, a timely turn to the "elimination" strategy may be necessary. . the "delay" group: all other countries/regions in fig. belong to the third group, which appears to follow the "delay" strategy, with # > , such that the epidemic will continue to grow. this is often referred to as the "flatten the curve" strategy, which aims not to reduce the epidemic to an as-low-as-possible level within a short period of time, but only to delay its growth through mediocre epidemiological control and social distancing measures. our results show that this is usually the worst scenario in terms of cost-effectiveness. a country/region may opt to this strategy because their tradeoff between short-term certainty (economic loss avoidance) and longterm uncertainty (possible disappearance of the epidemic in the summer, development of vaccines, etc.) leans towards the former. unless they have strong evidence to justify the tradeoff, we strongly suggest they reconsider. moreover, our results show possible directions to improve-enhancing the social interaction control for the infected population through more comprehensive testing or enhancing the social interaction for the exposed population through stricter social distancing measures, whichever sees fit based on the location of the country/region on fig. . rest of the world: more than countries/regions do not appear in fig. due to the lack of data, most of which are third-world countries/regions. although little information is available to us about the situations in these places, we conjecture that they may at this moment be pursuing cost-effectiveness of their antiepidemic interventions because of their limited availability of resources, which we call the "worth every penny" strategy. as our results show that the most cost-effective measures are usually neither the most effective one (actually they are usually very ineffective), nor the least costive ones, the "worth every penny" strategy is not a good option either. if a country/region opts to this scenario solely because of the lack of resources, it should be viewed as a humanitarian disaster, and we call for international aid in this situation. at the end, we acknowledge the extreme difficulty of even trying to lay out the comparison between human lives and economic activities, or the tradeoffs of lives between different social groups. we believe that the ethical discussion should be open to the whole society and hope that this article can contribute to the discussion. ( ) and a network transmission layer based on inter-city migration. through inter-city travel, the numbers of exposed and infected populations are adjusted daily. the model is calibrated using the migration data and the number of reported cases in china. see materials and methods for model specifications. (a) gradient of the first effectiveness function (with the number of infected cases as the metric for effectiveness) at the peak/inflecting stage, displaying decreasing margins, as well as the reverse of the effect of the inter-city travel level as ! and " decreases. (b) gradient of the second effectiveness function (with the number of cities with infected cases as the metric for effectiveness) at the accelerating stage, displaying firstly increasing and then decreasing margins as ! and " decreases. (c) gradient of the second effectiveness function (with the number of cities with infected cases as the metric for effectiveness) at the ending stage, displaying monotonically decreasing margins as each dot represents a country/region. the sizes of the dots indicate the number of infected cases on april , . classical seir model characterizes the dynamics of the susceptible, exposed, infected, and removed population, assuming a certain population size and transmission rate. in closer scrutiny, the fixed transmission rate assumption requires contact-based transmission process ( ) . from the perspective of spatial interaction, this requirement is contradictory to the prior assumption of fixed population. due to the high mobility of modern society, fixed population requires a large spatial scale of the model, e.g., the national scale. moreover, contact-based assumption is valid only at the scale of human daily activities-such scale should not exceed the urban scale to incorporate multiday intercity travels. at a larger spatial scale, human activity is featured by the "return-explorer" dichotomy ( ) . the former spreads outbreaks through daily contact, while the latter "diffuses" the epidemic through long-distance travels. the order of magnitude of the intercity long-distance migration can be massive in china, and related studies have also discussed the effect of this population flow on the spread of covid- outbreak ( , , , ) . thus, the covid- outbreak is actually subject to a "contact-spread" two-step transmission process. therefore, the classic seir model is insufficient to accurately describe the epidemic dynamics in the urban network and needs to be expanded. thereupon, we constructed a series of seir models for all cities, coupled with an intercity network. the node-edge structure of the model corresponds to the above-mentioned contactspread process; and the required data (epidemic parameters of each city, and intercity spatial interaction) are relatively simple. here, the choice of appropriate spatial resolution is critical. on the one hand, while prior research have adopted similar model design, they conducted the analysis mostly at the provincial resolution ( , , ) . according to the analysis above, the provincial level is not the best choice to reflect the intercity-spread mechanism. on the other hand, at too fine spatial scales, such as km-grid, township, or census blocks, modeling results can hardly be verified due to the lack of data, and random errors can be large. attempting to make a compromise between simplicity and completeness, interpretability and prediction accuracy, data spatial accuracy and verifiability ( ) ( ) ( ) , we took cities (administratively called prefectures in china) as the unit of analysis and built a model which we call the spatial-temporal explicit seir model (stex-seir). this model can not only be used to analyze the number of infected cases, spatial range of infection, and dynamics of the covid- outbreak, but can also be used to reveal the arrival time of the first case in each city. we calibrate the model with chinese case statistics. although it does not cover the entire globe, it is still informative to other countries since china has gone through the whole process of the epidemic. the stex-seir model relies on the following assumptions: ) intra-city population is homogeneous, i.e., it is a "meta-population," which is a common practice in the absence of real demographic data; ) omitting the impact of foreign imports and exports of covid- cases: the proportions of imported and exported cases during the covid- outbreak in china are extremely small and can be omitted over the study period. ) virus remains unmuted: although a very large number of mutations have been recorded ( ) , the transmission patterns of the virus have not noticeably changed so far. first step: update the immigrated and emigrated populations, the exposed population, and the infected population (assuming that the inputs and outputs of n, e, and i across cities are completed instantly at the beginning of time t). • ,-./ : lockdown on january ; • -*( -. : measures taken on january ; human-to-human transmission was confirmed on that day. : the proportion of the susceptible turning into the exposed status. second step: seir model in each city: in the above equations: s (unimmune and susceptible population) may turn into infected by contacting infected individuals; e (exposed) is the population who are in the incubation period after being infected; i (infected) is the population being symptomatic and infectious; r (removed) is the population who died or have been cured-they can neither infect others nor be infected again. n: total population in the city, subject to = + + + . other notations: • " : probability that a susceptible is infected and becomes an exposed person; • ! : probability that a susceptible is infected by an exposed person; • : probability that an exposed person turns into infectious status; • : probability of being cured; • : probability that an infected person dies; • " : probability that an exposed person is infectious; • ! : the number of susceptible persons who an infectious individual contact during each step of simulation; • " : the number of susceptible persons who an exposed individual contact during every step of simulation. the case data used in this article come from the national and provincial health commissions in china ( ) . wuhan is the city hit hardest by the epidemic, accounted for . % of the total number of cases. the number of cases in hubei province other than wuhan accounted for another . %. therefore, wuhan and hubei province have very different medical conditions, prevention and control measures from other cities and provinces in china. thus, we assigned different initial parameters for wuhan, other cities in hubei, and cities in other provinces. the initial number of cases in wuhan was set to be , and the initial numbers in other cities in hubei and in other provinces was set to be . according to the national report, the first confirmed infected case in wuhan can be traced back to december , . thus, we chose december , as the starting point for the simulation. according to other studies ( ) , this date is as close as possible to the real starting point of the pandemic, and epidemiological data are only available from this point to calibrate model. the endpoint of model calibration is february , when the epidemic in china was almost over and the model has converged by that time. we will simulate the spread of the covid- epidemic from that date until the end of april as verification and prediction (by the time of writing this article). we used chinese national, provincial, and municipal reports as the baseline to calibrate our model, so that the fitted number of cases would match with the surveyed data. on the one hand, the assumption, based on case statistics, is reliable. reports are released separately by more than cities nationwide and updated daily. panel data from different provinces are inter-validated with other and are also supported by data from other sources (e.g., total mortality rate of the population, or mortality data of similar symptoms such as influenza, pneumonia, etc.). thus, it is extremely unlikely that the data are fabricated continuously without being noticed. on the other hand, research shows that due to delayed diagnosis at the early stage of outbreak, the actual onset date of most confirmed cases is actually one week ahead of the report date ( , ) . considering the incubation period, we pushed the outbreak curve from the reported dates backwards by a few days, thus yielding the baseline for model calibration. the calculation and initial values of , , , , , , , " , ! , " , and ∆ were articulated in section of the supplementary material. among the three basic dimensions of epidemic control (control of the source of infection, cutting off transmission, and protection of the susceptible), we focus on the transmission cuttingoff dimension. specific measures belonging to this dimension can be further divided into three categories: ) "regular" epidemiological control measures; ) in-city social interaction control and ) intercity travel restrictions. these measures can be directly mapped to the three key parameters ! , " , and ∆ (denoted as tl henceforth) in the stex-seir model (table s ) , and thus empower the quantitatively evaluation of the effectiveness and cost-effectiveness of various scenarios. regular epidemiological control measures include thorough epidemiological surveys, close contact tracing, and quarantines of all the infected individuals and their close contacts as early and as possible-especially the close contacts, who may be potential carriers. these measures are reflected in the model by tuning the values of ! and " . it is noteworthy that the limits of the effectiveness of regular epidemiological control measures: • it is very difficult for the close-contact tracing and quarantines to be exhaustive, so they cannot reduce " to , indicating an effectiveness "roof"; • close contact tracing is a tedious work and is heavily manpower dependent. given that the epidemiological control system in regular practice usually has limited capacity. under outbreaks the system could be overloaded and becomes dysfunctional. under these circumstances, more aggressive measures are needed to reduce " . in-city social interaction control measures include work-from-home, shutdown of schools, workplaces, public transit, and public spaces, cancelation of public events, lockdown of residential neighborhood, etc. the effects of these measures are to reduce the chance of close contact between the infected/exposed and susceptible individuals, and are represented in the model by the values of ! and " , especially " , with contributions of different measures aggregated to a final value. we collected the measures adopted in different prior papers and coded them to estimate the final values of ! and " . it needs to be noted that the effects of specific measures are clearly overlapping with each other. for example, when social distancing both at the work and home ends are effectively implemented, the marginal "contribution" of an additional control on public transit usage would be minimal. also, despite of the nominal public orders, the actual stringency of the measures could be flexible in practice. for simplicity, we utilize expert knowledge to estimate the overall efforts, i.e., the values of ! and " . intercity travel restriction includes suspending inter-provincial and intercity buses, trains and flights, closing highways and roads, etc. the purpose is to reduce the intensity of personnel exchanges across cities, thereby reducing the transmission of the infected/exposed individuals. this is represented in the model by the reduction of spatial interaction parameter tl, or ∆ " and ∆ ! . in this paper we use two indicators to evaluate the effectiveness of disease control measures: the number of infected cases, and the spatial extent (represented by the number of cities with infected cases). minimizing the total infected population is a self-explanatory goal from the perspective of epidemic control. since e will eventually be converted to i with a fixed probability, we only need to focus on the infected population i and use the relative change of i over the simulation period (one month) as one indicator of effectiveness. the total antiepidemic effect in all cities is the summation: aside from the total number of infected cases, the spatial extent of the epidemic is also an indicator worth noticing. if the epidemic can be contained within a narrow spatial extent, the medical resources in other cities can be diverted to the infected cities to help, which actually happened in china where medical teams across the country were sent to wuhan for support. we also use the relative change of the number of cities with infected cases during the simulation period (one month) to quantify the effect: the total effect in all cities is the summation: we used the opportunity cost of economic output as the indicator for the cost of the antiepidemic measures. for simplicity, we only used the work-hour loss as a proxy for the economic output loss and did not convert it to monetary values of goods and services. travel restriction leads to reduction in the total number of personnel exchanges between cities. in particular, the outbreak of covid- in china overlapped with the spring festival holidays, and travel restriction apparently caused the migrant workers who previously travelled back hometown not be able to return to cities where they had worked after the holidays. while the impact on economic output is affected by factors such as the productivity difference between the migrant workers and local workers, we can use wages as a proxy for productivity and estimate the differences ( ) ( ) ( ) . calculations show that the average income of migrant workers in was . % of that of the local workers, or roughly the same, so we do not need to adjust for the differences in the model . therefore, this study ignores the role of the above factors. factors such as employment elasticity, which are more difficult to estimate, are also omitted for simplicity. therefore, the loss of economic output of city i at time t from travel restriction is: where _normal is the urban population in the normal status, approximated by the number of urban populations in the same period last year, and " _normal is the " in the normal status. for those who are not affected by the intercity travel restriction, we argue that the indiscriminately implemented in-city social interaction control measures reduce their intensity of social interaction, which in turn reduces their productivity and thus reduces the economic output. more specifically, we used " and ! as the proxy for productivity. it should be noted that lower " or ! is not necessarily the result of indiscriminate suppression of daily interactions but could also be the result of regular epidemiological control measures. a distinction needs to be made between these two situations. it can be reasonably assumed that when the number of the infected cases is small. for example, the daily increase of the number of infected cases does not exceed a certain threshold, the goal of reducing ! and " can be achieved solely by regular epidemiological control measures. therefore, a rational administration will choose not to opt to universal in-city social interaction control measures to avoid collateral loss. what is lost in this case is the output of only those who have been quarantined, i.e., the exposed and infected cases. we admit that it is extremely difficult to estimate such a threshold, which could be highly dependent on specific contexts such as governance capability, the resourcefulness of the epidemic control system, and cultural and geographic factors. although, a numerical estimation would still be of help. there was no opportunity to observe this threshold during the outbreak in china: in the early stage of the epidemic (before january , ), no effective epidemic control practices were taken outside of wuhan, and after january , the country had generally turned to aggressive and universal antiepidemic measures and it is difficult to separate the net effect of the each epidemic control measure. however, the singapore situation in january and february may constitute a good example, where in most cases daily reported new infected cases were under . another remark is that the chinese government sets a threshold of cases for reporting a newly discovered epidemic to the national cdc. in light of these numerical example, we use daily new infected cases as an estimate of the capacity threshold value. therefore, the loss of economic output of city i at time t from in-city social interaction control measures is set as follows. for straightforwardness, we convert the economic output loss into to a relative value: the proportion of the output with regard to that of the baseline (the normal situation). the total output loss ratio of city i during the entire simulation period is and the ratio of total output loss of all cities during the entire simulation period is: in summary, the comprehensive cost-effectiveness function of antiepidemic measures is: particularly, if cost = , it is stipulated that both cost/effectiveness ! , cost/effectiveness equal to . for simplicity, we only calculated the cost and cost-effectiveness functions at the national level (the entire city network). to discover any initial value-dependence of the cost and the costeffectiveness functions, we ran the simulation at different stages of the epidemic. based on the calibrated model, five scenarios with the following starting and ending dates are set: the model is run for days for each scenario, and five indicators are calculated: number of total infected cases, number of cities with infected cases, overall cost, effectiveness/cost ratio with respect to the number of infected cases, and effectiveness/cost ratio with respect to the number of cities with infected cases. we calculated the gradient function of the five indicators with respect to " , ! , and tl to analyze the structure of the solution space. for simplicity, we calculate the gradients discretely at the following points: ! = { , , , , , , , , , }; " = { , , , , , , , , }; tl = { , , , , }, where the five levels of tl correspond to . , . , . , . , and times of the normal intercity travel level. note that the lowest level of tl is not . this is because according to the situation in china (given by baidu migration data), when the tl is the lowest, the travel level is still about % of the normal level, and we believe that the stringency of intercity travel restriction in reality cannot be greater than this level. in contrast, aggressive in-city social interaction control measures can reduce the values of " and ! to levels very close to . we evaluated the antiepidemic situation of countries/regions based on the stringency of activity control policies of the infected and exposed, i.e., the degree of which ! and " are reduced. this takes steps: ) codifying policies and computation of the stringency indices; ) mapping of the stringency indices into values of ! and " ; and ) assessment of the antiepidemic situation based on the results of our analysis. for the exposed, effective activity control measures include close-contact tracing and social distancing. for the computation of the stringency of these measures, we used the data and method provided by the oxford covid- government response tracker ( ) , and computed and compiled the exposed activity control stringency index with six indicators: school shutdown, workplace shutdown, public events cancelation, public transport shutdown, public information campaign, and close-contact tracing. for the infected population, quarantine is the most important measure to reduce their activities, which but also requires testing in the first place. we therefore used the comprehensiveness of testing as the proxy for the infected activity control stringency. as testing policy coding from the aforementioned source is rather coarse, we instead designed an alternative index based on two indicators: tests per million population, and the ratio between cumulative cases and the number of total tests. the compiled index is gained through dividing the first indicator with the second, logarithmically transforming the quotient, and finally normalizing the result to the - range to match that of the exposed population's activity control stringency index. the logarithmic transformation is used only for visualization purposes, as very few countries/regions have much higher original indices. for the testing data, we employed sources from www.worldometers.info/coronavirus/. it should be noted that as mainland china does not publish testing data, we used the data from wuhan as a proxy. for both indices, we use the data as of april . a total of countries/regions are present in the final dataset. analysis results show that there exist two threshold values for both ! and " that correspond to the "elimination" (r << ) and "control" (r < ) strategies, as shown in table s . it should be noted that the effects of the two indicators are substitutable, with the elasticity of ! to be larger. for the mapping from ! to the respective stringency index of a specific measure, it is clear that the mapping should not be linear because of the non-linear transformations we have done. we therefore employed an estimation method based on expert knowledge: to achieve / reduction of ! which is required to "eliminate" the epidemic, it is needed to quarantine as many infected cases as possible. considering that about / to / infected cases are asymptomatic ( ) , this roughly means that all symptomatic cases should be tested. based on the statistics from wuhan and south korea, this requires the tests per million population to be greater than , , and the ratio between the number of cumulative cases and total tests to be smaller than %. converted from the above estimation to the respective stringency index, the value is around . we thus used this value as the boundary between "elimination" and "control". following similar methods, we estimated that the boundary between the "control" and "delay" policy groups to be around . for the mapping from " to the respective stringency index, because all six indicators' contribution to the actual stringency level are roughly equally weighted, we simply conducted a linear mapping between the composite stringency index and " . thus, the stringency index boundary between the "elimination" and "control" groups is , and that between "control" and "delay" is . epidemic parameter estimation is the first and fundamental step in the seir model. in previous studies, choices of these parameters usually adopted mathematical interpretation, such as the inverse of the incubation period or infectious period ( , ) and thus the numbers are not physically interpretable. however, if we consider the actual characteristics of covid- as shown in epidemiological studies, the ambiguity of the model can be greatly reduced. we estimated these parameters based on real-world data, considering errors caused by possible concealment, omission, and late reports. we used close contact tracing data after january to estimate ! , since statistical data in the early stage showed fluctuation due to the lag of reactions from the government. in the equation following, ( ) indicates the number of newly confirmed cases in a day. and ( ) indicates the number of close contacts who are still under medical observation in the same day. we averaged ! ( ) with regard to t and got . , so we set ! to %. in addition, we considered covid- to be infectious in the later stage of the incubation period: there is no evidence of weaker infectivity during the incubation period, and it is assumed that it can be as infectious as during the infected period, indicating " = ! = %. the incubation period is generally - days, and - days in most cases. the median incubation period is days. assuming infectivity in the - days before the end of the incubation period, with an average of days, the ratio of infectious exposed people ( " ) can be calculated as follows d is the average infectious day of the exposed, and e is the median of the incubation period. according to the new coronavirus pneumonia prevention and control program (second edition), suspected cases are those with similar clinical symptoms as infected people. if a suspected case tests positive for nucleic acid test or its viral gene sequencing is highly homologous with sars-cov- , it is diagnosed as a confirmed case. considering the relationship between suspected and confirmed cases, the ratio between new confirmed cases to existing suspected cases is recorded as the ratio of the exposed cases becoming infected cases. this index is a characteristic of the disease itself and is generally the inverse of the incubation period from the mathematical interpretation. therefore, it should be uniform for all cities. this parameter has converged since january and the average value is . ( . %). because of the presence of asymptomatic infections, only considering suspected cases can lead to overestimation. since asymptomatic infections accounted for . % of confirmed cases, we scaled up the suspected cases and the revised to be . %. according to the epidemiology working group of the chinese center for disease control, at least cases existed in hubei province in december , spread in fourteen counties. by january , , there were cases and deaths in cities of provinces. however, in the national epidemic reporting system, the first death was not reported until january in the whole country except wuhan. therefore, we took the results (table s ) published by the epidemiology working group and calculated the mortality rates in different regions at each stage (table s ) . we fitted the mortality rates with the following functions: wuhan: recovery rate is closely related to the capacity of local medical resources and city governments' responsiveness, so we conducted parameter fitting for each city. it is reasonable to believe that the number of cured cases published is reliable since there is no obvious reason for concealing recovery. therefore, the recovery rate is calculated as the ratio between newly cured cases and existed confirmed cases. the recovery rate has changed significantly by time. taking wuhan as an example, the abrupt outbreak led to a lack of medical resources at the beginning. then, aid resources from all over the world were sent to hubei province. in addition, huǒshénshān and léishénshān hospitals were built quickly to treat patients in severe conditions. these efforts contributed to the improvement of recovery rates ( ) . to capture the time-wise changes, we fitted piecewise functions to the recovery rates based on the characteristics of each stage. among the different stages, the early recovery rate in wuhan fluctuated too heavily to fit, so we took the average value in the period as an indicator of the recovery rate. the specific fitted functions are as follows: mainland china except hubei province: close contact tracing is a common epidemiological control measure, especially when early treatment is not clear, and when vaccines are not available. this approach can identify potentially infected individuals, quickly isolate them before they turn to severe patients, and prevent the occurrence of secondary transmission ( ) . social interaction intensities of the exposed and infected individuals are denoted as " and ! respectively in our model. these two parameters are highly related to local population densities. considering that the covid- outbreak occurred during the chinese new year, the intensity of social interaction was significantly higher than usual. wuhan, as china's large transportation hub, may have a higher per capita social interaction intensity than other cities. to address this, we used the contact rate of the infected population during sars (maximumly ) as the initial value of ! ( ). in general, the behavior of the exposed population is less restricted than the infected, so we assume that the maximum contact rate of the exposed is twice as high as that of the infected, which means that the maximum " is . according to the local epidemic reports, the initial values of ! and " in each area can be obtained (table s ) . then, based on the local control policies and response time, we adjusted the contact values at different stages. in january , , zhong nanshan announced that there was a risk of human-to-human transmission of covid- , and some people in wuhan began to consciously reduce their activities. until the citywide lockdown, the number of cases surged, and people began to realize the severity of the situation. then, strategies such as business shutdown, school shutdown, stay-at-home notices, and community shutdown were gradually implemented throughout the country to avoid contact between the susceptible and the infected population. in the late stage of the epidemic, numerous prevention and control strategies, including hospitalizing all confirmed cases and isolating all suspected cases, have greatly reduced the values of ! and " (figs. and ). the initial value of each city's population in our model is that on december . then, the population changed after the new year migration, which can be calculated through the spatial interaction matrix. our research includes prefectures in mainland china, with the time span from december , to april , . the data collected included migration data from the baidu migration map ( ), statistical yearbooks, and local covid- reports. the original baidu migration data is consisted of migration flows of , city pairs among cities from january , to february , . since there was no news of the covid- outbreak in december, the intensity of social activities was not affected and remained the same as normal workdays. in march and april after the outbreak, we assumed that due to the impact of the travel restrictions in most cities, residents' travel remained at a very low level. therefore, the migration data for december was set to be the same as an average workweek from january , to january , , and the intercity migration after february was set to be the same as between february to february . in addition, the numbers of covid- cases in our model and population data for the cities were collected from the national bureau of statistics of china ( ) and the national health commission of china's reports. we collected the publicly announced epidemic control policies of all prefectures in china from january to february . this dataset includes about , documents, among which were issued at the provincial level. we codified the policies into categories: one for longdistance travel restrictions, and the other five for in-city social interaction control measures (table s ) . we further translated the policies into their control stringency in terms of social interaction reduction based on expert evaluation on the policy terms as well as enforcement level inferred from media coverage. we lastly mapped the codified policies to the values of ! and " (figs. and ) . in the baseline (real-world) model, the initial number of cases at this stage was and increased to , after days. the initial number of infected cities were and increased to after days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is for ! and for " ), and that of ! higher when the level of social interactions is low (as low as ). however, the marginal contribution of ! and " are both - times higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ and " ≤ , the number of infected cases will shrink, and will be reduced to after approximately two months (table s ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high, and that of ! slightly higher when the level of social interactions is low. however, the marginal contribution of ! and " are both - times higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ and " ≤ , the number of cities with infected cases will shrink, and will be reduced to after approximately two months. since there was no baseline control at this stage, any change in the three coefficients means tighter control on social interactions, and hence a cost to economic output. first, the marginal contribution from the reduction of tl to the cost function is approximately - orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = , " = ) and ( ! = , " = ). the global gradient between the ridge and ( ! = , " = ) is moderate, and a lowest plateau appears when ! ≤ where the value of the cost function is . on the other side, the global gradient is much steeper, with a minimal value of the cost function that is also at the corner. the global cost peak appears at ( ! = , " = ), at which point the peak cost slightly exceeds % of the total output. in terms of cost-effectiveness, as the peaks and trends of the cost function and two effect functions are different, the two cost-effectiveness functions show greater non-linearity. in terms of the cost-effectiveness with respect to the reduction of the number of infected cases, two local peaks exist. one is at the "strictest control" point i.e., ( ! = , " = ), when each . % reduction in the number of infected cases is associated with % of economic output cost within a month; another peak occurs at the "non-control" point i.e., ( ! = , " = ), when each . % reduction in the number of infected cases is associated with % of economic output cost within a month. however, in the latter case, there will be no reduction in the number of infected cases at all according to the respective effectiveness function, while the former case coincides with the effectiveness peak. in terms of the cost-effectiveness with respect to the reduction of the number of cities with infected cases, situations are similar, only that a third peak appears at ( ! = , " = ), which is also a hardly effective solution. overall, the best solution considering both effectiveness and cost-effectiveness in whichever terms at this stage of epidemic is at the "strictest control" point (fig. s ). stage: january , to february , . in the baseline (real-world) model, the initial number of cases at this stage was , and increased to , after days. the initial number of infected cities were and increased to after days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is for ! and for " ), and that of ! higher when the level of social interactions is low (as low as ). however, the marginal contribution of ! and " are both up to two orders of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ and " ≤ , the number of infected cases will shrink, and will be reduced to after approximately two months (table s ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the patterns of marginal contribution of the three coefficients are similar with the "reduction of infected cases" case and we do not elaborate here. first, the marginal contribution from the reduction of tl to the cost function is approximately - orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = , " = ) and ( ! = , " = ). the global gradient between the ridge and ( ! = , " = ) is moderate and roughly linear, and a lowest plateau appears when ! ≤ and " ≤ (the "strictest control" scenario) where the cost is a small number ( %- %). on the other side, the global gradient is much steeper with increasing margins, with a minimal value of the cost close to at the corner (the "non-control scenario). one noteworthy character of the cost function is that the value of ! does not affect the position of the peak, but affects its value: keeping " the same, each % reduction in ! reduces the cost function by %- %. the global cost peak appears at ( ! = , " = ), or a "loose control" scenario, at which point the peak cost exceeds % of the total output. the intuition is that in this case, the control on social interactions is not sufficient to reverse the trend curve of the epidemic, and after the number of infected cases exceeding a certain threshold (the capacity "roof" of regular epidemiological measures to contain the epidemic), cities have to opt to a universal control on everyday social interactions to contain the epidemic, inflicting heavy loss on economic output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s . the baseline model witnessed the turning point of the epidemic, when the initial number of cases at this stage was , and reduced to , after days. the initial number of infected cities were and reduced to after days. in terms of the reduction of infected cases, the decreases of ! and " both have positive effects, but the effects are non-linear-with all returns diminishing marginally. the decrease of tl, however, have varying directions of effect dependent on the values of ! and " , with negative effect when both ! and " values are high and positive otherwise. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is for ! and for " ), and that of ! one order of magnitude higher when the level of social interactions is low (as low as ). however, the marginal contribution of ! and " are both up to two orders of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ and " ≤ , the number of infected cases will shrink, and will be reduced to after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve. and at the non-control scenario, the number of infected cases will grow to approximately , , or times the number at the beginning of the baseline scenario (table s ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the patterns of marginal contribution of the three coefficients are similar with the "reduction of infected cases" case and we do not elaborate here. first, the marginal contribution from the reduction of tl to the cost function is approximately - orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = , " = ) and ( ! = , " = ). the global gradient between the ridge and ( ! = , " = ) is moderate, and the lowest cost at the corner (the "strictest control" scenario) is a small number ( %- %). on the other side, the global gradient is much steeper, with a minimal value of the cost function close to at the corner (the "non-control scenario). again, the value of ! does not affect the position of the peak but affects its value: keeping " the same, each % reduction in ! reduces the cost function by %- %. the global cost peak appears at ( ! = , " = ), or a "loose control" scenario, at which point the peak cost exceeds % of the total output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s . in the baseline model, the initial number of cases at this stage was , and reduced to after days. the initial number of infected cities were and reduced to (wuhan) after days. in terms of the reduction of infected cases, the decreases of ! and " both have positive effects, but the effects are non-linear-with all returns diminishing marginally. the decrease of tl, however, have varying directions of effect dependent on the values of ! and " , with negative effect when both ! and " values are high and positive otherwise. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is for ! and for " ), and that of ! times higher when the level of social interactions is low (as low as ). however, the marginal contribution of ! and " are both up to order of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ and " ≤ , or ! ≤ and " ≤ , the number of infected cases will shrink, and will be reduced to after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve. and at the non-control scenario, the number of infected cases will grow to approximately , , or three times the number at the beginning of the baseline scenario (table s ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, with increasing returns marginally at most times except when the values of ! and " are both very low-a noteworthy character that is different with the symmetric accelerating stage. another noteworthy character of the effectiveness function is the occurrence of plateaus when ! ≤ , where the change of " and tl will not affect the spatial scope of the epidemic. first, the marginal contribution from the reduction of tl to the cost function is approximately - orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = , " = ) and ( ! = , " = ). the global gradient at both sides of the ridge drops close to with increasing margins and plateaus when ! is sufficiently low or high. again, the value of ! does not affect the position of the peak, but affects its value: keeping " the same, each % reduction in ! reduces the cost function by %- %, and when ! < , a plateau occurs where cost is minimal. the global cost peak appears at ( ! = , " = ), or a "loose control" scenario, at which point the peak cost exceeds % of the total output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s . in the baseline model, the initial number of cases at this stage was and reduced to after days. the initial number of infected cities were and reduced to (wuhan) after days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " about two times higher at all times. however, the marginal contribution of ! and " are both up to one order of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when " ≤ , or ! ≤ and " ≤ , the number of infected cases will shrink, and will be reduced to after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve (table s ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, this time all with decreasing returns marginally -a noteworthy character that is different with the symmetric early stage. plateaus also exist at this stage, when ( ! , " ) is to the left (smaller) side of ( , )-( , ), where any further change of the coefficients will not affect the spatial scope of the epidemic. at this stage, the shape and value of the cost function are both dominated by " . they decrease monotonically and roughly linearly along with the direction of " 's decline, and the effects of ! and tl are very small. at this stage, the epidemic at the baseline has come to an end. although the initial number of cases is roughly equivalent to the initial stage of the epidemic, the strict control at the previous stages has led to a significant reduction in the number of the exposed population. therefore, the cost function at this time is dominated by " , and the peak value of the cost function is only . % of the output, a great reduction compared to those in the previous three stages. since the lift of controls on social interactions will help restore economic output, the peak cost-effectiveness at this stage all occurs when all controls are lifted, at which time economic output will be very close to the normal level (when there is no epidemic). the second highest cost-effectiveness peak appears when the controls are maintained (given the number of infected cases, the controls now are realized predominately through effective epidemiological measures, rather than universal containment of social interactions), where the maximum loss of economic output is about . %. while the former appears tempting, in the long run (about two months later) it is likely to cause a rebound in the epidemic (we only run the simulation for one month, so this effect cannot be directly observed in the model. however, the situation is similar to the initial period of the epidemic, and thus the lift of controls is just equivalent to starting the entire epidemic from the beginning, such that a second outbreak is inevitable if other conditions remain unchanged). the latter, on contrary, appears to achieve a good balance between cost and effectiveness within this study's analytical framework (fig. s ) . table s . threshold values for ! and " of the "elimination" and "control" strategies. elimination (r << ) control (r < ) ! / of the normal value while " is the normal value / of the normal value while " is the normal value " / of the normal value while ! is the normal value / of the normal value while ! is the normal value world health organization, who director-general's opening remarks at the media briefing on covid- national health commission of the people's republic of china a new twenty-first century science for effective epidemic response early phylogenetic estimate of the effective reproduction number of sars-cov- the effect of travel restrictions on the spread of the novel coronavirus modified seir and ai prediction of the epidemics trend of covid- in china under public health interventions the lockdown of hubei province causing different transmission dynamics of the novel coronavirus ( -ncov) in wuhan and beijing the effect of human mobility and control measures on the covid- epidemic in china preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak estimating the value of containment strategies in delaying the arrival time of an influenza pandemic: a case study of travel restriction and patient isolation controlling pandemic flu: the value of international air travel restrictions fangcang shelter hospitals: a novel concept for responding to public health emergencies before wuhan lockdown, where did the , people go? variation in government responses to covid- version . cases and deaths by country, territory, or conveyance epidemic processes in complex networks returners and explorers dichotomy in human mobility covid- control in china during mass population movements at new year should internal migrants take full responsibility for spreading covid- modified seir and ai prediction of the epidemics trend of covid- in china under public health interventions effect of non-pharmaceutical interventions for containing the covid- outbreak in china a comprehensive framework for studying diffusion patterns of imported dengue with individual-based movement data impact of human mobility on the emergence of dengue epidemics in pakistan quantifying the impact of human mobility on malaria new crown pneumonia outbreak data sharing epidemiology working group for ncip epidemic response, the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china risk for transportation of novel coronavirus disease from wuhan to other cities in china why do wages increase with tenure ? on-the-job training and life-cycle wage growth observed within firms private-sector training and the earnings of young workers labor productivity: structural change and cyclical dynamics quantifying sars-cov- transmission suggests epidemic control with digital contact tracing key: cord- -zisirutu authors: mikalsen, marius; stray, viktoria; moe, nils brede; backer, idun title: shifting conceptualization of control in agile transformations date: - - journal: agile processes in software engineering and extreme programming - workshops doi: . / - - - - _ sha: doc_id: cord_uid: zisirutu agile transformation implies that organizations apply agile methods also outside of software development units. one particular way of doing such transformations is to create cross-functional software development units. this represents new challenges for control for organizations as the unformal agile control mechanisms from the software units meet the more formal, bureaucratic and hierarchical control from other units. the research on how to manage control in agile transformations, however, is scarce. through a case study of a new, cross-functional unit in a financial institution, we report on their work to implement control in agile transformations. to analyze our results, we draw on new perspectives for control in the digital era, which challenges existing presumptions on control. our findings indicate how agile transformations require rethinking traditional control mechanisms and experiment with new control perspectives more suitable for the digital era. the pressure of digitalization with rapidly changing markets and technology developments drive organizations towards adopting agile ways of working, also outside software development units [ ] . such agile transformation implies that agile methods are used not only in software development teams but also by other parts of the organization, such as business units [ ] . agile transformations deal with challenges such as hierarchical management in waterfall mode, difficulties working across organizational boundaries [ ] , and units not willing or able to change [ ] . one particular form of change aiming to overcome some of these challenges is creating semi-independent, cross-functional units (i.e. consisting of personnel from both business-and softwaredevelopment units) that use agile methods to improve the value of the software developed [ ] . collaboration across different units while working in new ways represent new challenges for control for organizations. the informal agile control mechanisms from the software units meet the more formal and hierarchical control from other units. how to implement control mechanisms that enable management to have control while still allowing the autonomy and rapid changes that are required in agile methods remains an open question in the literature of agile transformations. we, therefore, ask the following research question: how to manage control in agile transformations? to answer this research question, we report from a case study of a financial institution that implements a new semi-independent unit, an agile program, consisting of several cross-functional teams working according to agile principles. the teams consist of both software and business developers. in this paper, we report on their work on devising metrics for measuring the teams' performance using objectives and key results (okrs) [ ] . to analyze the results, we draw on new perspectives of control for the digital era, based on stewardship theory. stewardship argues that our conception of software development control needs to be reinvented in an era in which collaborative value creation is increasingly prevalent [ ] . our analysis indicates how agile transformations allow rethinking control and implementing new control perspectives more suitable for the digital era. the rest of the paper is organized as follows. section provides the theoretical background on how agile transformation challenges existing control, using stewardship theory as an alternative theoretical perspective. section introduces the case and explain how we collected and analyzed data. section shows the findings from one questionnaire and two retrospectives. in sect. , we discuss our findings in light of a stewardship perspective on control. section concludes and presents future work. agile transformation represents new challenges for control for organizations. previously, large software development projects were controlled by plans, hierarchies and standardization [ ] . as this is no longer suitable, new forms of control are introduced, such as large-scale agile frameworks like safe and spotify, with its own set of challenges [ ] that again can challenge autonomy [ , ] . in the digital era, managers face growing pressure to introduce techniques and practices to improve software productivity and reduce such impediments, however, it is unclear how impediments in software practices are controlled, i.e., identified, measured and managed [ ] . one technique many organizations use to guide and measure work is okrs [ ] . recent theoretical work suggests that we need new conceptualizations of control for the digital era. wiener et al. [ ] argue that the digital era, with increasingly advanced and pervasive technologies, changes how we develop software. changes include: new work practices with blurring of roles, a move from delivering commodities to continuous innovation, a move from hierarchical control structures to leveraging dynamic networks, and a changing workforce with increased specialization. previous research on control has had an agency perspective, which implied that the purpose of control is to ensure that project actors reduce self-interest and work according to a project, program or organization goal. now rather, the purpose of control shifts towards value creation, which has support in stewardship theory which considers the agent an intrinsically motivated steward working towards a common overall goal [ibid.]. wiener et al. [ ] outline key control questions and digital-era characteristics based on stewardship theory (see the themes of these two aspects in table below). first, key control questions are concerned with ) control modes, whether these are formal or informal, ) control style, and whether this is authoritative or enabling, and ) whether there is a value appropriation or value-creation purpose to control. second, digital era characteristics are concerned with: ) congruence with the common overall goal. ) information asymmetry is ok, ) intrinsically motivated actors, ) long term orientation rather than short term, and ) dynamic network structures. this study is a part of a longitudinal interpretative case study [ ] of agile autonomous teams set in a norwegian bank (dubbed norbank for anonymity), with more than , employees. norbank initiated in an agile program (ap) consisting of five crossfunctional autonomous teams organized in line with agile principles, with the goal of developing improved software for their business-to-business solutions in the insurance market. the teams consist of resources from both the software and business development side of the organization. the teams deliver software solutions to the business side of the organization, such as sales and settlements. the teams collaborate closely fig. . the participants discussing okr implementation with organizational units responsible for technology development and innovation. each team is led by product managers, who is part of the steering-forum of the program, together with managers from the business and technology units. the program has been developing software for a while and is now focusing on delivering value on the business side. their concern is how to measure and control these processes, while still being agile. in response, the program has decided to use okrs [ ] as a method for goal setting and measurement (fig. ). we collected data through two retrospectives with the program in february . the first retrospective was held with four product managers. the second retrospective was with the steering forum of the program. in this retrospective, a total of people participated. the participants included product managers, the leader of the program, data was collected by taking pictures, documenting the post-its on the whiteboards, taking notes, and collecting data through a questionnaire. our data analysis was partly deductive as we asked questions regarding stewardship theory in our questionnaire, but also inductive by analyzing our notes and pictures of the whiteboards for themes emerging in the retrospectives. we operationalized the control questions and digital-era characteristics from [ ] , as shown in table . the statements were given in norwegian and rated on a -point likert scale from strongly disagree ( ) to strongly agree ( ) . we collected the questionnaire responses digitally during the second retrospective, and the participants answered from their mobile phones. we decided that we wanted to collect the responses this way so that the participants could think individually in silence and also answer anonymously. furthermore, the tool we used (mentimeter.com) gave us the ability to show the answers to the questions in real-time, which sparked a discussion among the participants. as such, we were able to get feedback and better understand the responses. the participants scored the statements on a -point likert scale from strongly disagree to strongly agree. in fig. below, we see the score on key control questions regarding control configuration, enactment and purpose. team members control each other (item ) and have dialogue with those who decide the goals (item ). this is in line with a stewardship perspective on control. still, we see that traditional forms of measurement such as measuring on time and budget (item ) and what is produced (item ) are in place, indicating the remains of agency rather than value creation perspectives. measuring on cooperation between different actors is low (item ). in fig. below, regarding digital-era characteristics of control, we find scores indicating stewardship assumptions. we see a that practitioners are intrinsically motivated (item ). we see that their goals align with program goals (item ), that information asymmetry among actors is accepted (item ), and there is a lot of cooperation with people outside the team indicating dynamic networks (item ). finally, wee see that there is a is a long-term orientation in the work (item ), and a lower score on shortterm goals (item ). in the retrospective with the product managers, we found that there were collaboration and interdependencies with units outside the agile program. for example, developers in a separate software development unit made architecture decisions that led to the teams needing to rewrite their apis. also, they relied on developers from another unit on -strongly disagree implementing workflow automation, and these developers were often busy. moreover, collaboration with actors outside the organization was challenging. action items identified included increased use of okrs and continuous okr reviews as a way to focus the teams, the need for flexibility from the tech side on delivering on what the teams needed, and also get more competence on workflow optimization. in the retrospective with the program's steering forum, the participants discussed that the most important thing to improve was to better demonstrate to the business side the value of what the program delivered. the second most important was okrs and how this could be used to engage and involve the business side. the third most important was to keep the steering forum meetings lightweight and short. action items identified included: okr reviews, quarterly reviews with regard to overall goals, and clarify roles and responsibilities in the steering forum to make sure everyone could contribute more in future meetings. agile transformations involve that participants from different units, such as software and business, work together using agile methods [ , ] . such collaborations challenge traditional conceptions of control found in agile methods. to answer our research question -how to manage control in agile transformationswe have reported findings from a cross-functional unit in the midst of an agile transformation. we used the stewardship theory [ ] to shed light on how a cross-functional unit approach control. our results show that, regarding control configuration, enactment and purpose (fig. ) , team members control each other and have dialogue with those who decide the goals. this is in line with a stewardship perspective on control. still, we see that traditional forms of measurement such as measuring time, and budget and what is produced are in place, indicating the remains of agency rather than value creation perspectives. measuring cooperation is weaker and can be worth focusing on in order to manage known impediments for flow [ ] . regarding digital-era characteristics of control (fig. ) , we find strong indication of stewardship assumptions [ ] . practitioners are intrinsically motivated, their goals align with program goals, information asymmetry among actors is accepted, there is a lot of cooperation with people outside the team indicating dynamic networks, and there is a long-term orientation. this indicates that the ways of controlling in the agile program keep with the agile principles of mutual adjustment and autonomy [ ] . the retrospectives indicated an appreciation of using okrs as a way to set goals and communicate goals with the rest of the organization, such as the business units in particular, in order to ensure that what is valuable is agreed upon between units, and that value can be delivered. in terms of stewardship theory [ ] , such dynamic networks are a key characteristic of the digital era and should also be the focus of new forms of control. it is not clear exactly how to control it. in this case okrs are used as a way to communicate between the cross-functional teams and the business side. what is clear however, is that determining what is a valuable software deliverable will be an element of negotiation between interdependent units [ ] . in this paper, we have used stewardship theory to investigate how cross-functional teams work with okrs and how new forms of control can emerge in agile transformations. our results indicate that the members take responsibility for each other and that the team delivers; they also have an extensive dialogue with those who decide the goals. almost all stated that their goals aligned with the goal of the program, which indicates that the information flow and goal setting works well in this company. the participants also stated that it was ok that others had information that they did not possess. in terms of stewardship theory, this is called information asymmetry. stewardship assumes that knowledge workers are intrinsically motivated and selfactualizing and that they display a high level of commitment and involvement. the participants in our study reported being intrinsically motivated by working in the program. in sum, our findings indicate the balancing act between new and traditional control in agile transformations. a key limitation of this study is that it is a single case study. we find indications of stewardship assumptions in the case. however, how and what to measure and control remains a challenge during agile transformations. future research can investigate how for example, okrs can be used to support stewardship assumptions rather than older paradigms of control. also, old paradigms of control are not likely to disappear, so how to incorporate stewardship perspectives with other control regimes is relevant. in sum, agile transformation is about changing practices in organizations, and control seems to us to be a relevant aspect of changing practices. agile digital transformation: a case study of interdependencies understanding digital transformation: a review and a research agenda agile transformation: a summary and research agenda from the first international workshop moving is project control research into the digital era: the "why" of control and the concept of control purpose overview and guidance on agile development in large organizations implementing large-scale agile frameworks: challenges and recommendations networking in a largescale distributed agile project team autonomy in largescale agile the identification and classification of impediments in software flow a set of principles for conducting and evaluating interpretative field studies in information systems measure what matters: how google, bono, and the gates foundation rock the world with okrs ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use key: cord- -sxno tfd authors: gondim, joao a. m.; machado, larissa title: optimal quarantine strategies for the covid- pandemic in a population with a discrete age structure date: - - journal: nan doi: nan sha: doc_id: cord_uid: sxno tfd the goal of this work is to study the optimal controls for the covid- epidemic in brazil. we consider an age-structured seirq model with quarantine compartment, where the controls are the quarantine entrance parameters. we then compare the optimal controls for different quarantine lengths and distribution of the total control cost by assessing their respective reductions in deaths in comparison to the same period without quarantine. the best strategy provides a calendar of when to relax the isolation measures for each age group. finally, we analyse how a delay in the beginning of the quarantine affects this calendar by changing the initial conditions. abstract. the goal of this work is to study the optimal controls for the covid- epidemic in brazil. we consider an agestructured seirq model with quarantine compartment, where the controls are the quarantine entrance parameters. we then compare the optimal controls for different quarantine lengths and distribution of the total control cost by assessing their respective reductions in deaths in comparison to the same period without quarantine. the best strategy provides a calendar of when to relax the isolation measures for each age group. finally, we analyse how a delay in the beginning of the quarantine affects this calendar by changing the initial conditions. at the end of a novel coronavirus emerged in the city of wuhan, china. in january the disease was given the name covid- and, by mid february, china already faced over thousand cases [ ] . many scientists then began to model disease to forecast its worldwide impact ( [ ] , [ ] ), even influencing policies of many governments. as the disease spread across europe and the united states, some countries were forced to implement quarantines or even lockdowns to mitigate the harms and were able to do the so called "flattening of the curve", i.e., to postpone and dampen the maximum number of active cases. by mid may, brazil is entering this stage, with some states declaring stricter quarantine policies. optimal control theory has been applied to general epidemic models ( [ ] , [ ] ) as well as to specific disease models such as hiv ( [ ] , [ ] , [ ] ), tuberculosis ( [ ] , [ ] ) and influenza ( [ ] ). recently, a few works applying optimal control theory to the covid- pandemic, such as [ ] and [ ] , have appeared. this paper focuses on a seir model with quarantine as was proposed in [ ] , but dividing the population in age groups as in [ ] . this is particularly important since covid- has worse consequences on the elderly than it does on younger people. our goal is to calculate the optimal quarantine strategies numerically for different choices of parameters in the model, which reflect the decisions governments must make when implementing these policies, such as evaluating the economical costs of the quarantine for each of the age groups and when to start implementing the measures. then, we compare the controls by looking at how they reduce deaths in comparison to the same period without quarantine. the best strategy gave us a calendar of when to relax the measures in each of the age groups. our model consists of a classical seir model with a quarantined class. besides, we assume that the population has an age structure (see [ ] , [ ] for models with a continuous age structure and [ ] , [ ] for models with a discrete one). there are three age groups, described in table . and q i (t) be the number of susceptible, exposed, infected, recovered and quarantined individuals in each age group at time t ≥ , respectively. we assume that the total population all parameters are nonnegative. β ij is the transmission coefficient from age group i to age group j. typically, it will be assumed that β ij = β ji for all i, j. σ i and γ i are the latency and recovery periods, respectively, for age group i. λ is the exit rate from the quarantine. our controls are the u i (t), which denote the percentage of susceptible individuals in each age group that are put into quarantine at time t. as such, they satisfy, a priori, however, it is unrealistic to expect an entire population to stay under quarantine for a long time. there are essential workers such as healthcare professionals and police officers that cannot stay at home during these times. as most of these workers are in age group , we suppose that all of age groups and can be quarantined (for age group , indeed, this is especially important since they are a risk group for the covid- pandemic). thus, we shall loosen ( . ) by considering, for example, ≤ u (t) ≤ u max . evaluating u max is one of the tasks of each government's authorities. in this paper, we fix this parameter at u max = . . this means that be the total population of age group i. adding the equations in the system above, we see that n i (t) is also constant for i ∈ { , , }. since r i (t) only appears in the other equations as a part of n i (t), we substitute the equations for r i i(t) by n i (t).. hence, we may also consider the system of ( . ) parameter values are taken from [ ] and are listed in table . the data fitting was performed using an adaptation of a least-squares algorithm from [ ] . to see how the numbers of infections and recoveries are distributed in the three age groups, we refer to the data available in [ ] , shown in table . for simplification, we suppose that the difference between the number of cases and the number of deaths represents the number of table . parameter values (data from [ ] ). parameter value parameter value β . σ . recoveries. this is not necessarily correct, because some of the patients that we considered as recovered might still carry the disease, but we use this approach due to the scarcity of information regarding recoveries we currently have. the respective distributions are shown in table . according to [ ] , brazil had as of may , a total of , active covid- cases. even though there seems to be a large subnotification in the country [ ] , this number will be considered as the total number of infected individuals nonetheless. to estimate the number of exposed cases, we look at data from may , , since the mean incubation period of the disease is thought to be around days [ ] . once again according to [ ] , at this time brazil had , active cases, so this gives us an estimation of , exposed cases. we also suppose that these cases follow the age distributions of cases from table . finally, as of may , there were , recovered cases in brazil [ ] . therefore, our initial time will consist of data from brazil as of may , . the total population is assumed to be million, divided into % young people, % adults and % elderly. we also assume that there are no quarantined individuals when the simulation starts. since the numbers of exposed, infected and recovered are very small in comparison to the total population, we assume that the initial number of susceptible individuals is equal to the total population of the respective age group. the initial conditions of all variables, rounded to the nearest integers, are listed in table . using system ( . ), we consider the functional to be minimized as quarantined in the formula above, t is the quarantine duration and the parameters b i are the costs of the control. we assume that b i > for i ∈ { , , } and that where b ∈ r is the total control cost. sufficient conditions for the existence of the optimal controls follow from standard results from optimal control theory. for instance, we can use theorem . in [ ] to show that the optimal control exists. pontryagin's maximum principle ( [ ] , [ ] ) establish that optimal controls are solutions of the hamiltonian system with hamiltonian function where λ s i , λ e i , λ i i , λ q i and λ n i are the adjoint variables. these variables must satisfy the adjoint equations where i ∈ { , , } and c ∈ {s, e, i, q, n }. the adjoint system is detailed in ( . ) below. ( . ) the adjoint variables also must satisfy the transversality conditions finally, the optimality conditions come from solving this results in since we are considering bounded controls (because of ( . )), the u * i are calculated using where u max = u max = and u max = . . uniqueness of the optimal controls (at least for small enough t ) also follow from standard results, such as theorem . in [ ] . numerical solutions of systems ( . ) and ( . ) can be found by a forward-backward sweep method [ ] . the algorithm starts with an initial guess of the controls u , u and u and then solves ( . ) forward in time. after this first part, it uses the results and the initial guesses to solve ( . ) backward in time and new controls are defined following ( . ). this process continues until it converges. quarantines are not just a matter of public health, for they also present economic questions, for example. this means that we must pay close attention to the control costs b i . these numbers reflect how the population is capable of dealing with the quarantine of the respective age group. smaller values of b i mean that the population can withstand a stricter quarantine without many economical side effects. this is not possible, on the other hand, for bigger values of b i . since the bigger economic toll of the quarantine lies on the adults (because they form almost all of the economic active population), we assume that b is the greatest of the three values. as the isolation of the young implies closing schools, this educational impact makes b the second highest cost, albeit much smaller than b . how the total cost b is distributed among the age groups determines the shape of the optimal controls. to study this relationship, we let b = and define four cost distributions d , d , d and d , detailed in table . table . the four distributions considered for the total control b. distribution adults, then for the young and lastly for the elderly. the differences lie in the times it takes for these relaxations, which are described in table . our goal now is to compare these distributions by investigating the number of deaths caused by the pandemic at the end of the quarantine for each one of them. as in [ ] , the deaths will be calculated as a fraction of the recovered, since there is no disease induced mortality in our model. from table , we can derive the case fatality rates µ , µ and µ for age groups , and , respectively. the results are ( . ) = . , µ = = . , µ = = . . let d denote the number of deaths at the end of the quarantine due to the disease. by our discussion above, we can write because of the uncertain nature of the parameters and due to the high number of unreported cases, we do not show the crude numbers of d for the four distributions. the approach we use is to select the smallest values as unit and then scale the other values accordingly. the results are displayed in table . we also included d , representing the outcome of no quarantine. table . proportion of deaths in comparison to the lowest outcomes. on the one hand, we see that all the optimal controls reduces the number of deaths in more than times in comparison to "doing nothing" scenario. on the other hand, the four distributions produce very similar results, with d being slightly better for both quarantine lengths. for a better visualisation, we plot the graphs of the total number of infections for distribution d in figure . both curves reach their minimum around the same time that the controls reach zero. for a quarantine of days, the number of cases at the end is, indeed, much smaller than they are in the beginning. however, for a shorter quarantine, there could even be more cases than the initial total. this fact shows that quarantines cannot be too short, or else the overall situation in the end could be worse than in the beginning. moreover, the number of cases start to go up again towards the end. to finish this section, we analyse how the initial conditions affect the optimal control. we can interpret this as a way to see what happens if it takes too long for these measures to be implemented. we do this by considering initial conditions of exposed, infected and recovered twice and four times as much as their original values. as of may , , the number of active cases in brazil doubles every days [ ] , so this means waiting or days, respectively, to start the quarantine. in the plots of figure , we consider distribution d and quarantine length of days. the plot on the left of figure is the same as the last one in . the quarantine is relaxed for the young, adults and elderly after , and days, respectively. the plot in the middle has the initial conditions of exposed, infected and recovered multiplied by two. now, the quarantine is relaxed only after , and days, respectively. the last plot multiplies the initial conditions by four. the relaxations happen, respectively, after , and days. this shows that the quarantines have to be much stricter if there is a delay in their implementation. moreover, applying ( . ) with and without quarantine, we see that the deaths are reduced by factors of and when initial conditions are multiplied by and , respectively. with an earlier quarantine entrance, table showed that the reduction was by a factor of for distribution d , so the longer it takes for the quarantine to start, the less effective it is. in this paper we considered an age-structured seirq model, where the quarantine entrance parameters are thought as controls of the system, and we looked for the optimal controls via pontryagin's maximum principle. after writing down the optimality system, we calculated the optimal controls numerically and analysed how some of the parameters influence the results. these parameters represent the difficult choices authorities must make, such as deciding how many essential workers are allowed to remain circulating, estimating the economic impact of the quarantine and even when to start it. as such choices are made, the optimal controls give guidelines of how to proceed. in section , we considered a constant total control cost and distributed it among the age groups in four ways. the distribution with the best results with regard to deaths during the quarantine gave us a calendar of when to relax the isolation measures in the three age groups (for quarantine lengths of and days, respectively): • for the youngs, the date of relaxation was the th or the th day. • for the adults, the relaxation started at the th day or the th day. • for the elderly, it started at the th day or the th day. the optimal controls that induce this calendar produced a reduction in the number of deaths of and times, respectively, in comparison to the same period of time without quarantine. however, in both cases the number of infected cases reached a minimum just before the end of the simulation, so by the time the quarantine ended, the cases were going up again, even becoming bigger than the original values for the shorter length we considered. this shows that the quarantines are not effective if they are not long enough. we also showed that waiting too long to start the quarantine makes the period before the relaxation become longer. this also produced a loss in efficacy, since the reduction of deaths due to the quarantine (in comparison to the "doing nothing" scenario) appeared to be approximately inversely proportional to the number of initial cases. in our model we used data from brazil as initial conditions. brazil is a very large country, with many cities at different stages of the pandemic. this means that studies such as this one should be made locally to best suit the characteristics of each city. as the plots of figure suggest, the sooner the quarantine is implemented, the shortest the time the controls need to stay at their maximum is. optimal control of deterministic epidemics assessing the efficiency of different control strategies for the coronavirus (covid- ) epidemic enfermedad por el coronavirus (covid- ) optimal covid- epidemic control until vaccine deployment. medrxiv ( ) impact of nonpharmaceutical interventions (npis) to reduce covid mortality and healthcare demand optimizing chemotherapy in an hiv model optimal quarantine strategies for covid- control models mathematical analysis of an age-structured sir epidemic model with vertical transmission modeling the control of covid- : impact of policy interventions and meteorological factors optimal control of an hiv immunology model optimal control methods applied to disease models optimal control of treatments in a two-strain tuberculosis model optimal control of the chemotherapy of hiv the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application optimal control for pandemic influenza: the role of limited antiviral treatment and isolation optimal control applied to biological models an introduction to mathematical epidemiology optimal control of non-autonomous seirs models with vaccination and treatment mathematical theory of optimal processes using a delay adjusted case fatality ratio to estimate under reporting optimal control for a tuberculosis model with reinfection and post-exposure interventions disease extinction and disease persistence in age structured epidemic models nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study global dynamics of a discrete age-structured sir epidemic model with applications to measles vaccination strategies dynamics of a discrete age-structured sis models e-mail address: joao.gondim@ufrpe.br the authors would like to thank césar castilho (ufpe) for all the valuable discussions and suggestions during the preparation of this manuscript. key: cord- -jps j a authors: miranda, mary elizabeth g.; miranda, noel lee j. title: rabies prevention in asia: institutionalizing implementation capacities date: - - journal: rabies and rabies vaccines doi: . / - - - - _ sha: doc_id: cord_uid: jps j a rabies in asia and africa contributes to over % of human rabies deaths that occur in the world today. the vast majority or % of these deaths are in asia. practically, more than four billion people in asia or about % of the world’s population are at risk of getting rabies where an estimated % of documented human cases are from an infected dog bite. canine-mediated rabies is one of the few communicable diseases that can possibly be eliminated by currently available vaccines and tools for veterinary and public health interventions. with a more comprehensive and integrated approach, it is expected that dog rabies will be eliminated in target areas, and there will be an eventual decline and disappearance of human rabies cases. the burden of rabies is primarily on human health but the disease control has to be focused on the animal source. the ultimate goal of a truly regional disease program is to control and eliminate dog-mediated rabies and protect and maintain rabies-free areas in asia. current regional efforts aim to strengthen the intercountry coordination, and technical and institutional capacities to manage dog rabies elimination programs. the regional and national implementation efforts provide strategic direction and cooperation to ensure successful implementation of rabies control measures and eventual elimination. the focus areas include human rabies prevention through pre- and postexposure prophylaxis, mass dog vaccination, surveillance and epidemiology, laboratory diagnostic capability, public awareness and risk communication, legislation, dog population management, and establishment and protection of rabies-free zones/areas. existing mechanisms for implementation, when applied, give emphasis on one health collaborations. areas. existing mechanisms for implementation, when applied, give emphasis on one health collaborations. for most countries in asia, canine rabies is endemic and the majority of human rabies exposure results from dog bites particularly among children. the estimated number of human deaths across asia and africa is approximately , , with over . million disability-adjusted life years (dalys) and . billion usd economic losses annually [ ] . the vast majority of these deaths is in asia ( . %). india, with % of human rabies deaths, accounted for more deaths than any other country. rabies is often neglected when health and agriculture (animal health) agenda and budgets are set even if the costs and economic benefits have long been described [ , ] . it continues to be neglected and very often its public health impact is minimized by other priority infectious diseases like dengue, malaria, tuberculosis, and hiv. reliable data indicating the actual incidence of human rabies and rabies risk exposures are often lacking or non-existent in many countries, leading to the global number of human deaths that is significantly underreported [ ] [ ] [ ] . canine rabies is not only a major burden in endemic countries where thousands of human deaths occur annually, but also in previously rabies-free areas where risks of re-emergence have been increasing over the last decade [ ] [ ] [ ] [ ] [ ] . the burden of canine rabies is substantial, even though the disease is entirely preventable. dealing effectively with the problem is contingent on investing in the control at the animal source, which has long been lacking. long-term mass dog vaccination with high enough coverage could reduce health sector and societal costs with more rational and judicious use of postexposure vaccination [ , ] . disease elimination is feasible with currently available vaccines and disease control methods; however, innovative financing models are required to overcome institutional barriers. in , the first who interregional consultation on strategies for the control and elimination of rabies in asia laid down the impetus for many asian countries to promote and pursue the elimination of canine rabies to eventually eliminate the disease in human populations [ ] . asian countries were urged to develop comprehensive national plans with improved access to modern human vaccines and application of new economical postexposure treatments, better disease diagnosis and surveillance, and processing of data at the national, regional, and global levels, intersectoral collaborative efforts for dog rabies control and plans to expand public and health care worker awareness regarding rabies control and prevention. the association of southeast asian nations asean call for action toward the elimination of rabies in the asean member states and the plus three countries (china, japan, and korea) by demonstrated the key importance attached to rabies control at a political level [ ] . the asean rabies elimination strategy (ares) was developed in to provide a strategic framework for the reduction and ultimate eradication of canine rabies in asean member states. the strategy describes an integrated one health approach that brings together the necessary sociocultural, technical, organizational, and political pillars to address this challenge. the ares was designed to complement the existing subregional frameworks developed to control and eliminate human rabies, such as those developed by the asean expert group on communicable diseases (aegcd) in . in south asia, considering the importance of consolidating achievements in rabies control in member countries, the who regional office for southeast asia has developed a regional strategy for elimination of human rabies transmitted by dogs ( ) [ ] . in the middle east and central asia, human cases still occur, and dogs are the main vector. these regions plus countries of north africa and europe, belong to the middle east and eastern europe rabies expert bureau (meereb), an interregional rabies prevention and control network. in , meereb has called for elimination of dog-transmitted rabies through vaccine and rabies immunoglobulin stockpiles and implementation of a one health approach to achieve rabies eradication [ ] . across the continent, there is a marked increase in community-based initiatives for domestic animal vaccination and control with increased government support to funding and better program implementation. animal rabies control activities vary across the region [ ] [ ] [ ] [ ] . many national and subnational programs and demonstration projects have proven that proactive mass dog vaccination is much more effective at controlling rabies and less costly than campaigns that vaccinate in response to the occurrence of cases. control through proactive vaccination followed by two years of continuous monitoring and vaccination should be sufficient to guarantee elimination from any area not subject to repeat introductions [ ] . the degree of success of national and global canine rabies elimination efforts, however, depend heavily on effective epidemiological surveillance, which should ensure that intervention impacts can be monitored through time and outbreak responses initiated where necessary. it is recommended that rabies control programs ought to be able to maintain surveillance levels that detect at least % (and ideally %) of all cases to improve their prospects of eliminating rabies, and this can be achieved through greater intersectoral collaboration [ ] . rabies is a community-based problem that requires a well-organized and funded community-based approach. many countries need to strengthen their communitybased programs and implementation platforms, especially where government lacks the capacity and effective governance to mobilize community efforts [ , ] . in these settings, well-organized community efforts that aim to support or augment existing government rabies elimination programs are much desired. in countries such as indonesia, philippines, sri lanka, and thailand, these are often facilitated by nongovernment organizations and civil society organizations closely coordinating with the local government and private sector groups. there are practical bottom-up approaches that can be appreciated by governments. effective community-based approach seeks to strengthen the capacity of families, individuals, organizations, institutions, and systems to support disease programs and outbreak responses. it is expected to contribute in programming to reduce rabies risk and address community vulnerabilities, and enhance community and institutional resilience, being sensitive to the issues directly confronting communities, and desiring to support families and all sectors involved (whole-ofsociety) to take the necessary actions to reduce dog bites and rabies transmission risks. working through the lowest unit of a community, the family or household unit, is central, as "more resilient families are the foundation of more resilient communities." individual family units should be fully aware of rabies threats and the required interventions and be the first to take action when these threats appear, such as reporting dog bite incidents to community leaders. the ability of a nation to eliminate rabies starts at household levels-with family members understanding the risks of rabies and being able to systematically mitigate spread, and therefore ensure neighborhood rabies security. fostering private sector commitment to building and empowering communities through their corporate and human resource is another key element to effective community efforts. an integrated approach is the most effective way of protecting humans from canine rabies, as the infection is maintained in domestic dog populations [ ] . a number of countries have achieved considerable success in canine rabies elimination through mass dog vaccination. the feasibility and cost effectiveness of this approach have been strongly advocated in recent years, with major international public and animal health organizations declaring global canine rabies elimination as a realistic goal. significant progress in the production of rabies vaccines for human use that are low cost, rapidly immunogenic, safe and practical to use has led to increasing accessibility to timely and appropriate pep. in the early s, thailand pioneered the pep intradermal (id) regimens using cell culture-derived vaccines [ ] . from the time it was endorsed by who in , accessibility further improved due to increase in numbers of animal bite management centers, and better quality of services [ , ] . this eventually eased out the production of mammalian nerve tissuederived vaccines. in the philippines and sri lanka, since mid- s, animal bite treatment centers were established in government hospitals and major health facilities [ , ] . minimum essentials include training for mds and nurses in the proper management of patients and rabies exposures, cold chain, and systematic recordkeeping or registry. to date, id regimens are the first line in majority of the national rabies prophylaxis protocols and recommendations. rabies immunoglobulin of equine origin (erig) are in short supply throughout the world and particularly in asia, the demand is high [ ] . erig available in asia is either manufactured in europe, india, or china. though erig is considerably cheaper than human origin immune globulin, modern production of immune sera generates highly purified and safer products of better quality. producers of erig should be encouraged to continuously aim for consistent antibody levels and the least incidence of adverse reactions among patients. current recommendations for pre-exposure schedule use id injections of cell culture vaccines as a cost-reducing alternative for developing countries. as a strategy to augment human rabies prevention measures, childhood rabies immunization has been included in the national programs of countries like the philippines and vietnam. human vaccine production capacities of asian countries have improved greatly. rabies vaccine supplies come from a mix of private and public manufacturers in several asian counties and are mainly for domestic use, but some manufacturers have the potential to export vaccines. over the last decade, both private and public vaccine manufacturers in asia have exerted extra efforts to meet stricter government registration requirements as countries adhere to international and regional gmp (good manufacturing practice) standards and vie for who pre-qualification. the national regulatory authority (nra) in most countries generally enforces their local gmp standards, which tend to be stricter and more demanding to the producers [ ] . the procedure for approval of newly introduced vaccines is very much in place, involving the conduct of complete preclinical and clinical testing and establishing lot consistency prior to approval for marketing. most countries have functional nras/national control laboratories (ncls), which provide overall control on the vaccine production process and final product quality. exceptionally, a few countries still lack the capacity to perform rigid laboratory testing. demand and supply human rabies vaccine manufacturers are growing in number most markedly in china and india with more than manufacturers serving a combined population of more than billion with an estimated demand for rabies vaccines of about million doses or about million full-course treatments per year. the vaccine production levels typically range from , doses to million doses per year (for cell-based production facilities), with some producers in china and india upgrading their capacities to produce more [ ] . vaccine types china and india discontinued the production of mammalian nerve tissue origin (nto) vaccines in , and vietnam stopped producing the sucklingmouse brain vaccine, which has been in use over years, in [ ] . it now imports cell line-based vaccines, and they have also modernized their main vaccine manufacturing plant (gmp compliant) located in hanoi. all these manufacturers follow the who standard requirements, as well as refer to usp requirements. there are essentially types of modern vaccines, according to the substrate, that are produced mainly in china and india, namely the primary hamster kidney (phk) cell, vero cell, human diploid cell (mrc- ), embryonated duck egg, and chick embryo cell. of these, the vero cell is the only continuous (animal) cell line. all these vaccine types undergo concentration and purification processes by either zonal centrifugation or tangential filtration and gel chromatography. the virus strains being used include the pv (pasteur institute paris or cdc) and pm (wistar institute), and in china they also use their locally derived strains. vaccine preparations are either liquid or freeze-dried single dose ( . or . ml) in glass vials and are administered according to the essen regimen [ ] . quality asian vaccine manufacturers generally apply in-process control measures that include sterility tests, elisa, srd, and nih potency testing. national regulatory authorities only issue marketing licenses if complete testing of vaccines, including preclinical and clinical studies, has been conducted. laboratory testing regimens applied essentially follow who requirements, and lot release systems are being constantly reviewed and modified. in china, a system of random testing of production lots and post-market surveillance/testing and product recall are being strictly put in place. random lot testing generally includes tests for sterility, safety, and potency (nih method). most rabies vaccine manufacturers (public and private) seek to be who pre-qualified as they consider it advantageous to the marketing and worldwide distribution of their products [ ] . costs in china and india, the cost of modern locally produced vaccines range from to usd per dose. in china, imported vaccines are - usd per dose. in southeast asia, imported vaccines typically range from to usd per dose. hold backs and the way forward some countries that intend to start their local modern rabies vaccine production need support to establish cell line-based vaccine production, such as seed virus, cells, technology transfer, and funding for equipment or facilities. as who prequalification is sought for rabies vaccines, manufacturers observe that the process takes too long. as more new manufacturers join in, the demand for training of personnel on gmp must be addressed by all stakeholders. the reliability of the currently prescribed nih potency test is a major problem that manufacturers face in the production and control of rabies vaccines. essentially the nih test gives varying results depending on the laboratory and the status of the mice which the test utilizes. some manufacturers have also questioned the need to conduct the stability testing of vaccines on a per batch basis [ ] . in general, countries are able to follow the who requirements for human rabies vaccine production. the complete replacement of nto vaccine with cell line-based rabies vaccines has been accelerated, as india and china have demonstrated the feasibility of domestic commercial vaccine production. countries generally have the desire to produce better quality vaccines but are concerned about the effect on supply, and how to get the production of cell line-based vaccines started. regional supplies of relatively inexpensive vaccines will surely influence the decisions of countries to produce their own cell line-based vaccines. it would be advantageous to countries if who introduced a system of recognizing (qualifying) domestically produced vaccines using various types of cell substrate, and encourage exports where appropriate, principally to lower the world price of human rabies vaccines. to increase awareness and enhance community participation and support, public information and education are necessary. components of the information campaign generally have discussions on rabies as a fatal disease, its epidemiology, and its prevention and control, the disease control program in general and related national and local rabies ordinances as they support the program implementation and responsible pet ownership. with the realization of the impact of rabies in daily lives, and that pets can be a source of human infection, implementing community and schoolbased programs were relatively easy to roll out. volunteerism, active engagement, and willingness to pay of people in the program stems likewise from communitybased initiatives [ ] . community-based programs found all over asia concentrate on campaigns using multimedia (television, radio, newspapers, internet/mobile devices), display of posters and banners in strategic areas, distribution of flyers and other materials, public hearings of local ordinances and hosting of village assemblies. some educational campaigns are often conducted at various government offices and in churches or other religious structures. generally, celebrations like the world rabies day are observed to remind people of the continual threat of rabies and the importance of the program to control and eliminate the disease. school-based rabies educational programs, designed to improve awareness about rabies prevention among children are common in countries like india, philippines, thailand, and vietnam. these were mostly developed and are implemented with the ministry of education and in coordination with the ministries of health and agriculture. in the philippines, the integration of rabies education into the school curriculum was initially developed by the department of health's national rabies control program in [ ] . lesson plans prepared by school teachers integrated facts and figures about rabies, and lessons on responsible pet ownership. activities for the children involve fun educational events to celebrate the bond between children and pets. the power of the youth must be harnessed. lessons taught in interactive school programs could be brought into households and be ingrained in family values. rabies awareness in youth and adolescents will ripple through the entire family unit, thereby ensuring sustainable rabies interventions in future generations. the youth can proactively be involved in dog vaccination and control. governments that are committed to implementing disease control programs provide the institutional framework, legislation and policies, infrastructure and logistics, human resources, and budget appropriation. the legal framework for implementing rabies prevention and control programs is already in place in most countries in asia. national legislation defines the roles and responsibilities of the councils including dog and dog owner registration; collection of registration fees; animal population control; dog vaccination; surveillance of human and animal rabies and exposures; settlement of disputes/agreements between bite victims and dog owners; and promotion of responsible dog ownership. funds for disease control programs are traditionally sourced from local and national governments, and international development aid. actual implementation of intersectoral rabies control programs often requires and depends on regular budget allocation as mandated by law. international aid agencies and nonprofit organizations offer funding and technical inputs, pooling of resources, set guidelines, and standards, have monitoring and evaluation mechanisms and often act as an intermediary between donors and government. in any disease control program, wide stakeholders' involvement is critical [ ] . it is important to bring together key stakeholders from business and the public sector to discuss health security and the importance of establishing public-private partnerships. contributions from private organizations, including businesses, academe, and civil society, can be tangible and intangible. tangible efforts are generally in the form of donations in kind or money. intangibles such as voluntary efforts should be maximized. the intense involvement of the local communities has served as a conduit for business sectors, nongovernment organizations, academic institutions, and civil society organizations to extend their financial and technical assistance to the government. the national government agencies can sustain the standardized approaches to rabies control and elimination and promote how to start the public-private partnership that would ensure sustained intervention. such technical and administrative conduits are essential and beneficial to all stakeholders, providing the credibility and quality assurance that is directly rooted in the day-today field operations. there are numerous examples of public-private partnerships that contribute to public program implementations, support research and promote policy development in bali, indonesia, india, sri lanka, philippines, thailand, and vietnam [ ] . a number of rabies control programs in humans and animals have sourced funds from different sectors at different levels. the range of sources could be from the grassroots to the corporate and people's organizations. general support to local governments given by partner organizations includes community mobilization, volunteer services, and materials donations. the business sector gives direct donations or embarks in joint ventures. the academe conducts research and offers technical inputs, voluntary services, and student manpower. the community contributes taxes, fees for service, donations, and volunteer manpower. field implementers and partner communities often face constraints such as high operational cost, wide regions of coverage and labor intensity. many innovative approaches have been attempted to overcome these problems. there are numerous lessons of good practices learnt from experience. an example of a successful, sustainable community-based integrated rabies control program is the bohol rabies elimination program, implemented as a partnership between the provincial government, the national government line agencies (health, agriculture, education, interior, and local government) and a few nonprofit organizations. the project brought together educators, physicians, veterinarians, government officials, community leaders and the general public, and aligned them for coordinated effort [ ] . this program produced a significant shift in rabies control, from government-dependent implementation to a community-led movement. collateral benefits included better conditions for animal welfare, more responsible pet ownership, and improved public safety. ownership of the program at the community level has assured more engaged field operations and sustainability. attaining the goal of rabies control and eventual freedom from disease became a shared concern. there are challenges though to public-private partnerships. the continued assurance of private-sourced funds depends on the effort to acquire these; thus, fund sourcing must be a full-time effort that requires a wide range of committed stakeholders. the credibility that has been established through successful local programs facilitates fund sourcing. field experience showed that there could be disincentives to provision of external assistance including an uncertain political environment, lack of political support, and inadequate counterpart funds [ , ] . the key to the success of a public-private partnership model is the strategic partnership among the community-based stakeholders with sound technical and operational capabilities to implement the rabies control and elimination program framework and strategic plan. the partnership ensures evidence-based and informed program planning, institutionalized organization, policies, and implementation mechanisms, the setting in place of clear performance indicators, and uninterrupted resource inputs. the key steps in project integration within the local system is the identification of key persons or technical and political champions, clear and functional feedback channels among partners (e.g., internal and external monitoring), and encouraging government empowerment and program ownership, stakeholder participation and formally defining roles and responsibilities of stakeholders through memoranda of understanding. increased public awareness and understanding enhance willingness to pay and contribute for public good. program sustainability is a critically important issue for all public health programs, but especially for resource-poor countries with limited budgets and many problems to resolve. thus, a successful rabies prevention and control program must be built around integration and the strengthening of intersectoral and transdisciplinary collaboration and cooperation between several societal components [ , , ] . the asean rabies elimination strategy gives particular importance to the organizational and one health framework for rabies elimination [ ] . as an example to understand better, the expansiveness of one health challenges: in dealing with urban rabies threats, it is recognized that the best single approach is to attack the disease at its source, that is, to eliminate dog-mediated rabies. eliminating dog rabies greatly reduces the need for postexposure human prophylaxis, at least at some point in time if the process is executed systematically. in this regard, the health sector has been at the forefront of rabies elimination programs. while this traditional principle of rabies elimination is proven to be one of the most well-based and sound of disease control strategies, in reality program implementations are confounded with complexities, resulting in more failures than successes (with only a few established and emerging exemptions). the failures have often been associated with the re-emergence of rabies after it had been temporarily eliminated in the dog population. even areas (e.g., islands) once rabies-free have encountered emergence and endemic spread of urban rabies [ ] [ ] [ ] . this has been the general situation for many decades. while the prescribed solution is sound and tested, i.e., elimination of rabies at-source, in the overall process, whole-of-society must deal with the complexities of prevailing urban rabies. detailed scientific argument is not necessary to point out that poverty is a strong driver of rabies endemicity. for example, the massive proliferation of slum areas is directly proportional to rabies proliferation. the survival priorities of people dictate their health and wellbeing-seeking behaviors; obviously, food and shelter come first to those who are hungry and cold. in the same way, hungry stray dogs seek food and shelter, and the proliferations of garbage and market wastes drive these behaviors. populations, whose general health and wellbeing are deteriorating, will be further drawn into the state of poverty. where there are people who (must) eat dogs, there will be those who propagate and market dogs legally or illegally. there are a number of undesirable reasons why dogs are able to cross boundaries and islands. and there will always be bad governance that reciprocate bad community participation/cooperation. such complexities are too numerous to mention all here but are at the heart of why programs fail. very similar arguments also apply to the continued proliferation and emergence of other infectious diseases [ ] . most significantly, poverty dynamics clearly drive vulnerabilities to diseases [ ] [ ] [ ] , and these include ( ) lack of adequate safe food and water; ( ) lack of protection from harm such as exposure to pests, inclement weather, pollution, violence, stress, and disasters; ( ) extreme social marginalization and deprivation of opportunities to earn a living, to be educated, to receive healthcare; and ( ) infliction of collateral harm, especially to woman and children, the disabled and the elderly. clearly, the determinants of infectious diseases are multifaceted and increasingly complex [ ] . poverty reduction is central, as generally, poverty alleviation means vulnerability reduction. this has been documented in relation to the likelihood of infectious diseases emergence in impoverished community settings [ ] . good governance, involving the highest inter-ministerial central body for one health coordination backed up by legislation and a clear mandate, budget appropriation, resources mobilization, and pilots or model programs that lead to policy development, provide optimism to implementing comprehensive operational plans that are vertical and horizontal, national and sub-national. these are important institutional drivers and enablers for a sustainable public-private partnership. comprehensive rabies control programs should consider combining human, financial, and material resources with other interdisciplinary disease programs to benefit from synergy and maximization of shared resources. with the guidance of oie, fao, and who, governments, donors, foundations, and other private partners should be mobilized to sustain investment in canine rabies control and eventual elimination. pursuing the regional goal of rabies elimination cannot be taken lightly. sustained investment mechanism and integrative efforts must be enabled, for instance, by the designation of a specifically mandated body, e.g., a rabies or one health authority directly under the office of the president or prime minister. such body could be assigned a czar (secretary or minister level) and a dedicated budget for office and resources. it should be solely focused on rabies elimination (in the meantime), and collaborate as necessary with the health, veterinary, education, environment, industry, and other sectors on clearly defined parameters and terms, with its authority maintained at all levels, i.e., national to local. the structure and mechanism for this could be legislated. such legislation, together with the creation of the one health authority, will remain relevant to the continuous prevention, control, and eradication of any zoonoses threat (e.g., ebola, influenza, sars, mers-cov, malaria, leptospirosis) that potentially are pandemic threats. it is important to recognize the main justification for these radical recommendations which is: any country with a prevailing human rabies threat in this modern and highly connected world is considered a hindrance to global progress. all stakeholders are specifically drawn to the enhancement of governance. this is to ensure a sustainable approach to comprehensive capacity strengthening and broader risk reduction in the context of community resilience and regional security. the overriding objective is to advocate for continued and better targeted funding to strengthen capacities to immediately and effectively detect, prevent, and prepare for and respond to any infectious disease/zoonosis outbreaks and similar major threats. targeted initiatives must promote broad resilience objectives, cognizant that absolute efficiency of systems, especially in relation to widespread threats, is contingent on the interdependencies of sectoral and systems approaches, and the capacity to enable strategic systems synergies. whole-of-government/whole-of-society coordination, involving multi-sectors within communities, is key. therefore, rabies and zoonoses preparedness needs to be integrated into emergency and crisis response systems. the systematic involvement of even the military should be pursued. the one health authority's core structure, functions, and capacities to plan, prepare, mitigate risk, and respond to threats through its dedicated rapid response teams should be sustainable. these must integrate into the broader whole-of-society platform and proposed "one resilience" approach [ ] to effective interactions among national and regional entities involved in the prevention and control of rabies and other zoonoses (depicted in fig. ) , to the extent that all actors understand their roles and are enabled to effectively respond when major threats strike, so that normal operations, economic activities, and livelihood are protected and sustained. estimating the global burden of endemic canine rabies rabies control in the republic of philippines: benefits and costs of elimination re-evaluating the burden of rabies in africa and asia review of rabies epidemiology and control in south, south east and east asia: past, present and prospects for elimination basel: karger surveillance guidelines for disease elimination: a case study of canine rabies the rabies epidemic on flores island, indonesia ( - ) re-emergence of rabies in dogs and other domestic animals in eastern bhutan epidemiological and clinical features of human rabies cases in bali history of rabies control in taiwan and china one health: the theory and practice of integrated health approaches. wallingford: cabi towards canine rabies elimination in cebu, philippines: assessment of health 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elimination project renewed global partnerships and redesigned roadmaps for rabies prevention and control integrative societal resilience or 'one resilience' approach: towards optimal health and wellbeing infections and inequalities: the modern plagues sustained global attention to emerging pandemic threats and risks: the need to strengthen one health systems and whole-of-society preparedness global report for research on infectious diseases of poverty. special programme for research and training in tropical diseases social determinants of infectious diseases in south asia ochungo p mapping of poverty and likely zoonoses hotspots. dfid zoonoses report biosecurity within one resilience. paper presented at the asean regional forum (arf) cross-sectoral security cooperation on bio-preparedness and disaster response workshop key: cord- -chwk bs authors: nan title: abstracts: poster session date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: chwk bs nan an immune etiology has been postulated for acute cerebellar ataxia of childhood (acac) since it frequently follows viral infections. we analyzed serum and cerebrospinal fluid (csf) from acac patients for antibody cross-reacting with cerebellar neurons. serum and csf were obtained within days of onset of pancerebellar ataxia from subjects aged . to years. varicella infection preceded cases. results of enhanced cranial ct scans were normal; csf demonstrated - cells/mm with sterile cultures. serial dilutions from : of serum and undiluted csf were screened for antineuronal antibody by indirect immunofluorescence (iif) using frozen, unfixed normal human cerebellum. serum ( : ) was examined further for antineural antibody by western immunoblotting using purified cerebellar neuronal extracts as antigen. serum from age-matched, neurologically normal pediatric inpatients served as the control group for iif and immunoblot experiments. in acac patients, no antineuronal immunoreactivity was observed by iif. immunoblots demonstrated no consistent pattern of immunoreaction when comparing acac to controls, though patient exhibited distinct bands at kd (neurofilament protein) and kd. although antecedent infection suggests an immune etiology for acac, our preliminary results do not support a humoral mechanism for this disorder. ingrid taff; joseph zito, robert gould, and steven pavlakis, great neck and manhasset, ny in a -month period we studied patients between the ages of weeks and years with magnetic resonance angiography (mra). studies were performed on a . t magnet (siemens magnetom sp) with a circular polarized head coil. a three-dimensional time-of-flight technique was utilized. occasionally, images were obtained after gadopenetate dimeglumine infusion. two-dimensional projection images were calculated using a maximum intensity projection algorithm and recorded on laser film. sixty-seven patients also had routine mri. a sampling of vascular lesions was demonstrated. nineteen patients had clinical and mri evidence of stroke. mra revealed intracranial vascular occlusion in patients, diminished focal cerebral flow in the affected area in , and generalized ipsilateral underdeveloped cerebral circulation in . a moya-moya vascular pattern was found in and sickle-cell vasculopathy was found in patient. seven mras were normal. seventeen vascular hamartomas were demonstrated including vene of galen malformations, arteriovascular malformations, and venous angiomas. three aneurysms were found. thirty-one mras were normal. we find m u to be a valuable adjunct to routine mr imaging in the evaluation of pediatric patients with potential cerebrovascular disease. it demonstrates a spectrum of pathology, is noninvasive, and allows for serial follow-up examinations. angiography in pediatric cerebrovascular disease p . thalamic change in acute encephalopathy of adult rats. twelve weeks after grafting, clinical and histological studies were performed. we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. the survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin section. animals that received the transplants demonstrated qualitative and quantitative improvements in several parameters of locomotion. donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. we suggest embryonic rat spinal cord transplantation may be a useful treatment of spinal cord injury and a possible therapeutic strategy in human spinal cord injury and amyotrophic lateral sclerosis. the basic neuropathophysiology of hemineglect after unilateral cerebral lesions is still not clear. one theory holds that degraded perceptual processing occurs in the damaged hemisphere due to intrahemispheric deficits. another holds interhemispheric interaction at fault, with the intact hemisphere actively inhibiting spatial cognitive processes in the damaged one. we tested adult macaca fascicuhris with acute neglect on a task in which the whole visual surround was restricted to degrees from central fixation, and a second in which an opaque lens occluded the eye either ipsilateral (ipsi) or contralateral (contra) to the lesion. using paired t tests, in the first task there were no differences in reaction time to the ipsi and contralesional hemifields. in the second, there was no change in extent of the ipsilesional field (obtained with the contralesional eye occluded), as compared to its extent without occlusion. the contralesional field, however, improved significantly ( p < . ) with the ipsilesional eye occluded. since reducing sensory input to both hemispheres leads to no worsening of hemineglect, but reducing sensory input to the intact hemisphere alone leads to improvement of hemineglect, we conclude that adverse interhemispheric interactions play a major role in the pathophysiology of hemineglect. we assessed the sensitivity and applicability of a new, cornbined cognitive and mood screening battery for multiple sclerosis (ms). sixty consecutive, untreated clinically active ms patients, each relapsing-remitting and chronic progressive, underwent the battery and head mri upon entering concurrent treatment trials. the battery combines the faust-fogel brief cognitive screen and visual analogue dysphoria scale, both previously validated in other neurological diseases. cognitive domains tested were immediate and delayed sentence and word-pair recall, verbal fluency, and conflicting response suppression. patients marked ''usual mood" along a "happy-sad" cartoon continuum. relapsing patients were program and abstracts, american neurological association younger ( . vs . yr mean), with shorter ms durations ( . vs . yr), and had lower kurtzke disability scores ( . vs . ). modified qualitative mri grading (lesion burden, confluence, localization) was compared. half as many relapsing patients ( % vs %) scored ''abnormal'' cognitively, despite similar "sadness" rates ( % vs %). subjective dys-~ phoria in both groups correlated with denser periventricular lesion burdens. the battery was well tolerated and easily administered within minutes without special equipment. this combined cognitive and mood screening battery is sensitive and convenient for clinically active ms. alternate forms of the battery are needed for repeatability. questionnaires may be reliable and valid supplements to laboratory tests for brain-damaged patients, as they can be applied to situations for which laboratory testing is not possible. we investigated the usefulness of informant-based data in alzheimer's disease (ad) by comparing caregivers' subjective evaluations of probable a d patients' performance on an abbreviated version of the memory self-report questionnaire to objective evaluations derived from an extensive battery of neuropsychological tests and to clinicians' evaluations. similar information was obtained from healthy agematched controls. caregivers' subjective appraisals of patients' memory correlated significantly with objective measures of secondary memory, with all cognitive variables, measures of activities of daily living, and clinicians' evaluations of dementia staging. scores were independent of clinical indicators of depression. the abbreviated memory questionnaire showed good reliability, internal consistency, and external validity. its positive predictive value is . and its negative predictive value is close to %. results suggest that ( ) informant-based questionnaires may be useful for obtaining valid information on cognitive ability outside of laboratory settings; ( ) the scale reflected more than just memory functions; and ( ) the scale may be promising for screening cognitive difficulties in epidemiological or clinical settings. although neglect along the horizontal dimensions of extrapersonal space is well recognized, there are only a limited number of observations documenting neglect along the vertical and radial spatial dimensions. we report an investigation of neglect along the principal dimensions of extrapersonal space in a patient with bilateral mesial temporo-occipital infarctions. neglect was assessed by asking the patient and controls to bisect lines of lengths oriented in directions with respect to the body: horizontal, vertical, and radial. our patient showed significant neglect of upper vertical and far radial space, as well as neglect of left hemispace. his line bisection errors were consistently in a direction opposite the slight directional biases shown by controls for all line orientations ( p < . ). the magnitude of the patient's bisection errors increased by moving the lines toward the neglected sectors of -dimensional space. neglect of upper vertical and far radial space was also evident on line cancellation tasks. our results suggest that following focal brain injury, neglect may be observed along all dimensions of extrapersonal space. these findings provide further empirical support for functional specialization within inferior and mesial temporooccipital regions for attending to upper vertical and far visual space (previc, ) . p . posterior cortical atrophy: degenerative disease with primary visuospatial and visuosemantic deficits a. kertesz, m . polk, and a. kirk, london, ontario, and saskatoon, saskatchewan, canada posterior cortical atrophy is a recent, and heidenhahn's disease is an old, label for a miscellaneous group of patients with imaging or pathological and clinical evidence of visuocognitive deficits and cortical atrophy localized to the posterior cortex. the extent of this cortical localization and the nature of the pathological findings are not fully agreed upon, but spongiform degeneration and alzheimer pathology have been described. detailed examination of patients who are representative of the problem and have uniquely specific deficits is presented. one patient had visual associative agnosia, prosopagnosia, and transcortical sensory aphasia. lexicosemantic experiments of categorization, word retrieval, and comprehension of auditory and visual stimuli showed a specific impairment of visuoverbal semantics. a striking preservation of phonological, orthographic and visual structural input, and intercategory dissociations was demonstrated. consistency of errors argued for specific loss of semantic knowledge. another patient with apraxia, primary visuospatial deficit, agraphia, and amnesia at the beginning had predominantly right-sided posterior cortical atrophy, demonstrating further fractionation of the entity and the striking specificity of visuospatial function. the behavioral specification of degenerative disease is clinically and theoretically important. permanent neurological deficits after ischemic stroke are mainly determined by the location and size of the infarct. clinical recovery also depends on the functional state of adjacent brain tissue, where both neuronal loss and deactivation without gross morphological damage may affect flow and metabolism to a varying degree (g. mies et al, stroke ; - ) , and where the ability to respond to stimulation by appropriate neuronal recruitment may be impaired. therefore, degree of resting hypometabolism and of responsiveness to functional activation may provide a measure of prognosis. in patients (age . . yr) with aphasia consequent to ischemic stroke of the dominant hemisphere, regional cerebral metabolic rate of glucose (rcmrgi) was measured at rest and in of them also during spontaneous speech, using positron emission tomography (pet) of -(f )-fluoro- -deoxy-~-glucose (fdg). the pet study and a standardized neuropsychological test battery to assess the main aspects of language were performed around the fourteenth day after the stroke, and the language functions were assessed again to months later. performances in various dimensions of language weeks and to months after stroke were related to rcmrgl in topographically meaningful areas at rest and during activation using wilcoxon-rank program and abstracts, american neurological association sum test and multiple regression analysis. severity of aphasia was assessed by the token test, which showed a bimodal distribution to slight and severe, and a lower representation of moderate cases. global and all regional cmrgl at rest and during activation were significantly correlated to scores in token test at first and second examination, with the highest correlation coefficients ( - . to - . ) for broca's, wernicke's, and left temporoparietal regions. for performance after to months, the relationships were still significant with lower coefficients. verbal fluency also was correlated to kmrgi, but with lower coefficients that slightly increased for the recovery state. language performance at different stages in the course after ischemic stroke was significantly related (r = . for token test, r = . for verbal fluency). however, there exists a high variability in recovery that may be explained by stepwise regression of metabolic values. significant effects were observed only for cmrgl of the left hemisphere outside the infarct (partial r' = . ) at rest and for cmrgl within the infarct ( . ), the contralat-era mirror region ( . ), and broca's region ( . ) during activation, with a sum of all partial weight factors of . at rest and . during activation. our results furnish indicators for recovery of aphasia: the resting metabolism of the left hemisphere outside the infarct, and the activated metabolism in residual tissue within the infarct and in languagerelated areas. although the hemispheric metabolism at rest might be related to neuronal loss and thereby to the brain's reserve capacity, the extent of metabolic activation indicates neuronal recruitment and the capability of neuronal networks for functional recovery. heparin therapy for acute myocardial infarction: the timi-i pilot and randomized trial combined experience m . a. sloan, t . r. price, m . l. tevrin, and s. forman for the timi investigators, baltimore, m d of , myocardial infarction (mi) patients treated with rt-pa and heparin, ( . %) developed ischemic cerebral infarcts (ci). all ci patients had detailed neurological evaluations and ( %) had c t scans. age range was to years (mean yr), were male, and were caucasian. electrocardiographic location of mi was anterior in ( %) and nonanterior in ( %). six cis occurred within hours; between and hours; between and hours; between and hours; during the second week; and others distributed over the weeks after study entry. six of cis did not involve cerebral cortex; ( %) had multiple cis. of cis thought to be embolic in origin, had at least cardiac abnormality (mural clot, wall motion abnormality, aneurysm, or transient atrial fibrillation) known to be associated more specifically with embolism than just the diagnosis of myocardial infarction. eight of ( %) with ct scans had hemorrhagic conversion of varying degrees. the time of occurrence and sites of ci after rt-pa and heparin therapy for acute mi are similar to those reported in the prethrombolytic area. nancy futrell andjeanne m. riddle, detroit, m i photochemical irradiation of the carotid artery of rats has been used to induce endothelial damage, producing a nonocc h i v e thrombus (that apparently embolizes spontaneously) thrombi and emboli and multiple cerebral infarcts. evidence for embolism generally has a presumptive component. to document further that cerebral infarcts in this model are indeed due to embolism, we studied the ultrastructure of the carotid thrombi and the presumed cerebral emboli using scanning and transmission electron microscopy (sem, tem). the right carotid artery of wistar rats was irradiated with a laser ( nm, mw/cm , min) following the injection of the photosensitizing dye photofrin , . mgikg. rats were sacrificed from to hours later. endothelial damage with formation of a fragmenting thrombus, composed mainly of platelets and erythrocytes (with no fibrin in most areas), was present in the carotid arteries of all rats by sem. sem was done on cerebral vessels, containing peripheral blood elements, with single ( ) and aggregated ( ) platelets (causing occlusion in ), single ( ) and aggregated ( ) erythrocytes (without occlusion), and single ( ) and aggregated ( ) leukocytes (without occlusion). tem demonstrated that the platelet aggregates did not adhere to the cerebral endothelium. the endothelial surface of all cerebral vessels was normal, which provided additional evidence that the mechanism of cerebral infarction in this model is embolism. model of repetitive ischemia: this effect is significantly enhanced when combined with mild hypothermia ashfaq shuaib and elisabeth sechocka, saskatoon, saskatchewan. canada there is considerable evidence that glutamate release resulting in activation of postsynaptic receptors (especially nmethyhaspartate) is a major mechanism of ischemic neuronal injury. in vivo experiments have shown that a more severe release of glutamate may be responsible for the excessive damage seen with repeated ischemic insults. we have shown that in cell cultures the effect of brief repeated insults is more severe than a single insult of similar duration. in the present study, we tested the protective effects of cgs- in a cell culture model of single ischemic and multiple-insult paradigm. in the multiple-insult paradigm, in some cultures cgs- was combined with mild hypothermia to see if this would offer additional protection. cgs- offered a dose-dependent protection in cell cultures exposed to a single ischemic insult. cgs- was protective to cultures exposed to repeated ischemic insults. the protective effects were enhanced significantly when they were combined with hypothermia, resulting in almost complete protection of the cultures. the combination of therapies appears to be a valuable strategy in neuronal protection during cerebral ischemia. toby i. gropen, i . prohovnik, t . k. tatemirhi, z. sharif; and m. hirano, new york, n y although a rare syndrome, mitochondria encephalomyopathy, lactic acidosis, and stroke (melas) may offer a unique insight into stroke mechanisms. we report novel observations in a patient with melas studied with serial and quantitative cerebral perfusion after stroke using """tc-ceretec spect and ' ixe rcbf. a -year-old man with melas presented with left-sided headache, generalized seizures, fluent aphasia, and right hemianopia. serial ct and mri showed infarction of the posterior left hemisphere in a multiterritorial distribution. spect performed days after stroke showed to % greater flow in the infarct than in normal brain, which reversed days after stroke. quantitative rcbf (m isi, reflecting mostly gray matter), when corpatients died. we recommend ct evaluation in all patients who have a seizure or lose consciousness during the peripar-tum period. despite intensive management, mortality is high. seizures should not be attributed to eclampsia without careful neurological assessment. when prenatal care is sought, women should be counseled about the dangers of cocaine to themselves as well as to their babies. changes in circulating blood volume following stephan a. mayer, matthew e. fink, laura lenniban, louise m. klebanoff; auis beckford, lsak prohounik, william young, and robert a. solomon, new york, n y reduction of blood volume (bv) has been implicated as a risk factor for delayed cerebral ischemia (dci) due to vasospasm after aneurysmal subarachnoid hemorrhage (sah). volume expansion guided by target filling pressures has gained popularity as a means of preventing or reversing dci; however, the adequacy of central venous pressure (cvp) as a reflection of bv in this setting remains unclear. we measured bv and cvp concurrently in patients ( males, females; mean age yr) day after craniotomy (mean . days after sah) and an average of . days later. the mean bv (mllkg) measured using chromium '-labeled red blood cells (rbcs) fell from . to . (normal range - ), a reduction of % ( p = . , paired student's t test). despite this, mean cvp (mm hg) remained unchanged ( . vs . ). similar reductions of plasma volume ( %) and rbc volume ( %) accounted for no change in mean hematocrit ( . vs . ). bv fell . % among grade iiiliv patients (n = ) compared to . % among grade / patients (n = ). a moderate correlation between bv and cvp ( r = . , p = . ) was found only with the first set of measurements. time-related alterations in venous capacitance, myocardial contractility, or systemic vascular resistance may explain our findings. axel rosengart, louis r. caplan, michael s. pessin, atherostenosis of the extracranial vertebral artery (ec-va) has rarely been studied systematically in series of patients with acute vertebrobasilar strokes or transient ischemic attacks. we identified by conventional angiography and neuroimaging (ct, mri, ultrasound, mr angiography) patients with ec-va disease among patients with posterior circulation ischemia. patients with cardiac sources of emboli were excluded. the probable etiologic mechanisms were: group a: vertebral artery origin (vao) atherostenosis with embolism- patients; group b: vao atherostenosis with hemodynamic spells- patients; group c: patient with vao atherostenosis and both intra-arterial embolism and hernodynamic spells; group d: ec-va dissection- patients ( unilateral, bilateral); patient had perioperative compromise of the ec-va with presumed intra-arterial embolism; group e: vao disease in addition to other distal vascular lesions- patients ( with intracranial va and with basilar artery [baf occlusive disease). ec-va disease is not always benign. vao atherostenosis and dissection of the ec-va are sources of intra-arterial emboli. hemodynamicrelated ischemia occurs with bilateral and unilateral va disease but is often transient. vao atherostenosis is often accompanied by severe occlusive disease of the intracranial va and ba. program and abstracts, american neurological association sebastian e. ameriso, vicky l. y. wong, andvas gruber, hidemi ishii, and mark fisher, los angeles and la jolla, c a , and kanagawa, japan hemostasis abnormalities are associated with ischemic stroke. these changes typically are demonstrated in antecubital venous blood samples and may not necessarily represent changes within the vasculature of the brain. the purpose of this study was to identify potential differences in hemostatic profile from samples of cranial versus noncranial venous sites in patients with acute ischemic stroke. eight patients were studied within days of acute brain infarction. some patients were studied on separate days. blood was drawn from the external jugular vein and immediately thereafter from an antecubital vein without the use of tourniquet. we measured hematocrit, leukocyte count, platelet count, fibrin d-dimer (cross-linked fibrin fragment), plasminogen activator inhibitor- (pai- , an important antifibrinolytic protein), and anticoagulant proteins thrombomodulin and activated protein c. a jugular-to-antecubital ratio was calculated for each paired blood sampling. thirteen paired samples were obtained from the eight patients. external jugular-to-antecubital ratios (mean to-antecubitat ratio for pal was significantly different from ( p < . ), with higher concentrations in jugular samples. in conclusion, levels of hemostatic proteins measured from cranial venous blood may differ from antecubital samples in patients with acute ischemic stroke. in animal models of transient cerebral ischemia, the effects of repetitive insults are more severe than a single ischemic episode of similar duration. we used the cell culture model of ischemia to determine if the effects of repetitive ischemia are similarly more severe in this model of ischemia. for cell culture, we used fetal mice cortical astrocytic and postnatal cerebellar (glutamatergic) granular neurons and cerebral gamma-aminobutyric acid (gaba)ergic cells. lactic dehydrogenase (ldh) (activity per gram protein) release in the medium was used as a measure of cellular damage. compared to a single insult, there was a large increase in ldh release during repetitive ischemia in astrocytes ( vs , p < . ) and granular cells ( vs , p < . ) (highly significant) and a modest (but significant) increase in the cortical neurons ( . vs . p = . ). the demonstration that repetitive ischemia produces more severe damage in cell culture would suggest that the mechanisms are not predominantly vascular. cell culture could prove useful to study the mechanisms of neuronal damage with repetitive ischemia. we studied the spontaneous recovery of neurological function after acute ischemic stroke using a standardized stroke nih stroke scale scale ( n i h stroke scale) to assess the extent of improvement, differences in stroke types, and early predictors of later outcome. we performed serial neurological assessments on admission; , , and hours after admission; and to days and > days after admission. twenty-six patients had presumed embolic occlusion of the middle cerebral artery (mca) and had a clinical diagnosis of lacune. admission score was better in the lacune group compared to the mca group. the mean scores for all patients improved by the to -day and the > -day examination, but the degree of improvement was greater in the mca group than in the lacune group at > days ( p < . ). the degree of change at to days correlated with the change in score at hours ( r = . , p < . ) and hours ( r = . , p < . ). most patients improve after acute ischemic stroke, but to variable degrees and at different rates. david w. desmond, thomas k. tatemichi, miguel figueroa, dew'itt t . cross, and yaakov stern, new yovk, n y to investigate the effects of lacunar infarction (li) on cognitive function, we examined li patients months after stroke (age = . ? . yr; education = . . yr) and stroke-free nondemented control subjects (age = . k . yr; education = . k . yr) with a battery of neuropsychological tests. li was defined as a presenting infarct of cc and a mean volume of any additional subcortical infarctions of cc on ct scan. using multiple regression analyses, with significance set at p < . to minimize the risk of type i error, we considered the role of li as a correlate of performance in multiple cognitive domains. controlling for the effects of demographic factors, vascular risk factors, alcohol use, and depression within the multivariate models, li was a significant independent correlate of deficits in memory (( = -. , p = . ), verbal (p = -. , p = . ), visuospatial (p = -. , p < .oool), abstract reasoning (p = -. , p = . ), and attentional skills (p = -. , p < . ). we further investigated the effects of infarct number, volume, and location, as well as atrophy, on global cognitive function within the li group. the only significant independent correlate of global cognitive performance was a preponderance of left-hemisphere infarctions (p = -. , p = , ). these results suggest that li may produce dysfunction in multiple cognitive domains, particularly when the left hemisphere is differentially involved. p . increased intracranial atherosclerotic stroke in hispanics and blacks from northern manhattan ralph l. sacco, christina zamanillo, t . shi, andj. p. mobr, new york, ny intracranial atherosclerosis has been found to be more frequent in blacks compared to whites, whereas hispanics have rarely been characterized. among consecutive patients from northern manhattan over age hospitalized at the presbyterian hospital from to , cerebral infarction occurred in whites, blacks, and hispanics. all patients had at least one c t scan, % had duplex doppler, % transcranial doppler, and % angiography. strokes were classified as atherosclerotic (ath), cardioembolism, lacunar, and as infarcts of undetermined cause. ath was further subdivided into extracranial (eath) or intracranial (iath) . overall, the frequency of ath was similar in the three racelethnic groups (white , black ' , hispanic ) . the distribution of the atherosclerosis, however, was different in whites compared to blacks and hispanics. whites had more eath stroke than blacks and hispanics (white %, black % , hispanic lo%), while iath was similar in blacks and hispanics and greater than in whites (white , black , hispanic %). nonwhites have more iath stroke than whites. the similarity in the distribution of atherosclerosis between blacks and hispanics argues for shared environmental risk factors, rather than genetic differences. ethnic differences in stroke risk factors may help explain differences in infarct subtype. we studied mild to moderate alzheimer's disease (ad) patients with a series of " water bolus positron emission tomographic (pet) activation studies, and compared them to similar studies in age-matched normal controls. for each group, pet images were mapped onto the subjects' mri scan, and results of a particular activation condition were averaged across the group. naming a series of pictures (line drawings of animals) minus counting abstract designs as a baseline produced strong activation of the anterior cingulate gyrus only in the ad group. silent reading of words minus viewing a baseline series of "xs" similarly showed strong activation of the anterior cingulate gyms in the ad subjects but not the normals. naming block (activation condition) of % unnamed pictures, minus a second block (baseline) of easily named pictures, demonstrated much greater cingulate activation in the ad patients, for naming of the more difficult pictures. we conclude that this cingulate activation may reflect the greater involvement of an attentional network (of which the anterior cingulate is a part) in tasks requiring a higher degree of "mental work" on the part of ad patients. dementia in alzheimer's disease j. w. pettegrew, k. panchalingam, w. e. klunk, and r. j alzheimer's disease (ad) predominantly affects the brain, resulting in the loss of multiple cognitive abilities. some studies suggest the membranes of peripheral cells are involved in the disease. to investigate erythrocyte membrane molecular dynamics in ad patients and age-matched controls, we investigated erythrocyte membrane molecular motion at the surface (fluorescamine), aqueous-hydrocarbon interface (dppe-ans), and hydrocarbon core ( j-as; ppc-dph) by steady-state fluorescence anisotropy measurements of probable ad patients ( males; females) and ( males; females) age-matched controls. cognitive function was assessed by the mini-mental, mattis, and blessed scales. we found that intergroup comparisons revealed decreased motion at the surface ( p = . ) and aqueous-hydrocarbon interface ( p = . ) and increased motion in the hydrocarbon core ( p = . ) of the moderately to severely impaired ad patients compared to the controls. in the ad patients, there were significant correlations between decreasing membrane surface motion and worsening blessed scores (males p = . ; r = . ; femalesp = . ; r = . ). these findings suggest that molecules are being produced in the brain of ad patients that gain access to the circulation. these molecules insert into the erythrocyte membrane and secondarily alter erythrocyte membrane molecular motion. the production of these molecules correlates with the dementia and could contribute to the molecular pathophysiology of the disease. parkinson's disease (pd) and alzheimer's disease (ad) are common disorders of old age and may therefore coexist. the prognosis in demented pd patients is poor and early recognition of such cases is therefore desirable. the objective of this study was to identify characteristics that distinguish pd + ad from pd patients during early stage. all patients were clinically evaluated over a -year period . clinical diagnosis of dementia was made only when unequivocal clinical evidence of progressive decline in memory and cognitive function was documented, and pathological diagnosis of ad and pd was made using standard criteria. twentysix patients who had only pd or pd + ad were identified; had no dementia and at autopsy had pd. six patients had clinical evidence of parkinsonism and dementia and at autopsy had distinct pathological findings-pd and ad. these cases could be classified as having simultaneous or sequential evolution of pd + ad. those with sequential onset had pd before age years but were inexplicably functionally disabled early on, whereas those with simultaneous onset manifested pd after age years. pd + ad patients had rapid disease progression, shorter survival, poorer drug response, and more side effects of levodopa than pd patients. to study the prevalence ofwhite matter lesions in the general elderly population, and to investigate whether white matter lesions were relatively frequent in subjects with classic vascular risk factors and with hemostatic risk factors, magnetic resonance scans were obtained of participants, aged to years, of the rotterdam elderly study. the subjects for the imaging study were a random sample from the general population, stratified by age and gender. t -weighted images were obtained in the axial plane. white matter lesions were considered present when moderate or severe periventricular hyperintensities or when more than small focal lesions or focal confluent lesions were found. overall, % of subjects had white matter lesions. the prevalence and severity of le-program and abstracts, american neurological association sions increased with age. history of stroke or myocardial infarction, presence of peripheral arterial disease, factor viic activity, and fibrinogen level were each significantly and independently associated with the presence of white matter lesions. significant relations with actual systolic as well as diastolic blood pressure, with a history of hypertension, and with plasma cholesterol were observed only for subjects between and years. this study suggests that white matter lesions in the elderly may be related not only to the classic cardiovascular risk factors. but also to hemostatic factors. joan m. swearer, paula nelligan, hanno muelher, beatrice woodward, and david drachman, worcester, ma although behavioral disturbances occur frequently in alzheimer's disease and other dementing disorders, little is known about the factors that predict their development or predispose to their occurrence. in the present study we examined sets of possible predictive/predisposing factors retrospectively for behavioral disturbances in mildly to severely demented, community-dwelling patients. the factors examined included: individual distinguishing features (age, gender, age of onset, premorbid personality traits, prior psychiatric history) and dementia severity (dependence in activities of daily living [adls) and self-care, duration of dementia, global disease severity). spearman correlations and t tests were used to assess the relative influence of these factors on the occurrence of types of aberrant behaviors: aggressive behaviors, disordered ideation, and motor abnormalities. forty percent of the patients exhibited aggressive behaviors, % exhibited disordered ideation, and % had motor abnormalities. neither a prior history of psychiatric disorders nor premorbid personality traits were associated with the occurrence of the target behaviors. dependence in adls and self-care and greater global severity were associated ( p < . ) with the frequency and severity of aggressive behaviors, disordered ideation, and motor abnormalities. these results suggest that severity of dementia is a consistent and reliable factor in the development of aberrant behaviors, whereas preexisting personality traits are not. dementia of the alzheimer t y p e w. j. burke, a. ranno, w. h . roccafrte, s. p. wengel. b. l. bayer, and n . k. willcockson, omaha, ne l-deprenyl is an irreversible inhibitor of mao-b that has been reported to cause modest improvements in short-term memory and behavioral symptoms in persons with dementia of the alzheimer type (dat). thirty-eight subjects meeting research criteria for mild d a t were enrolled in a placebo-controlled, double-blind trial of r-deprenyl at a dose of mg twice a day. subjects underwent extensive clinical and neuropsychological assessments at entry, and at and months. after months, subjects taking both l-deprenyl and placebo showed a significant decline in their scores on the mini-mental state examination, the clinical dementia rating (cdr) scale, and the sum-of-boxes score derived from the cdr. when the change in scores on these clinical measures was examined across the groups, there was no significant difference. there were no significant differences within or between groups on several behavioral measures including the brief psychiatric rating scale and the cornell rating scale for depression in dementia. neuropsychological testing demonstrated no significant differences berween groups based on mean score change. l-deprenyl did not affect cognition or behavioral symptoms of dat in this -month study. k. marder, m-x. tang, r. ottman, l. cote, y. stern, and r. mayeux, new york, n y the etiology of dementia in parkinson's disease (pd) is probably multifactorial but there may be a shared susceptibility for p d and alzheimer's disease (ad). reliable risk factor interviews were conducted with informants of nondemented p d patients (pd-d) and demented p d patients (pd + d) enrolled in a longitudinal community study of pd. p d + d were older ( . yr) than pd-d ( . yr) and had later age at onset of motor signs ( . yr) than pd-d ( . yr) ( p < , ). the frequency of smoking, alcohol use, head injury, and family history (fh) of pd did not differ but fh of ad was significantly more frequent in the pd + d group (or . , ci . - . ). using stepwise logistic regression, only age of onset of motor signs (or . ), education < years (or . ), and the interaction of age of onset of motor signs and fh of a d (or . ) were independent predictors of dementia in pd. to address variable years at risk for development of dementia, life table analysis revealed the cumulative risk of a d to age in first-degree relatives of p d + d was . , and . in pd-d relatives ( p < . ). cox proportional hazards analysis controlling for the differences in ages of the relatives of both groups yielded a rate ratio of . (ci . - . ) for the development of a d among p d + d compared to pd-d relatives. we conclude that a genetic susceptibility to a d may raise the risk for dementia in patients with pd. differentiated from alzheimer's disease? john c. mowis, elizabeth grant, rita canfield, eugene rubin, and daniel mckeel, jr, st louis. mo vascular dementia (vd) is believed to account for to % of all us cases of dementia; however, pathologically confirmed cases are quite rare. this discrepancy suggests that current diagnostic criteria lead to the clinical overdiagnosis of vd. twenty v d subjects (mean age . yr; men, women) were diagnosed solely on the basis of the presence of dementia, a history of stroke(s), and a documented relationship of stroke to onset andlor course of dementia; ischemic scores (is) and neuroradiographic findings were not used for diagnosis. compared with subjects (mean age . yr; men, women) with dementia of the alzheimer type (dat), there were no significant group differences for comparable clinical dementia rating stages of dementia for measures of language, activities of daily living, or general cognition. the vd group scored significantly higher than the dat group on the modified is (f [ , ] = . , p < , ). all autopsied d a t subjects had verified alzheimer's disease (ad); also had cerebral infarctions. the autopsied v d subjects had , , and cc of brain tissue affected by stroke; ( cc) also satisfied histological criteria for ad. we conclude that ( ) the clinical features of vd and a d overlap considerably; ( ) diagnostic criteria based on the temporal association of stroke with dementia may have predictive value for vd; and ( ) the frequent coexistence of a d and strokes indicates that refinement of criteria is needed to distinguish "mixed" and "pure" vd. clinicopathological correlation remains essential for any study of putative vd. left-handedness has been proposed as a marker for decreased survival in the general population, but possible effects of handedness on longevity in alzheimer's disease (ad) have not been examined. we hypothesized that left-handed ad patients would evince more rapid deterioration and therefore die at an earlier age than right-handed patients. subjects were demented patients consecutively confirmed at autopsy to meet nincds-adrda criteria for "definite" ad. handedness was determined from structured interviews with primary caregivers and validated for most subjects with the edinburgh inventory of handedness. age at onset of dementia symptoms retrospectively determined by caregivers was used to calculate the duration of illness at the time of death. because of reported gender differences with regard to longevity, we first partialled out effects of gender before using hierarchical regression procedures to test the hypothesis. four of men and of women with definite ad were left-handed. the mean age at onset did not differ significantly between handedness groups (f [ l,loo] = . ), but the mean duration of symptoms ( alterations in the optical properties of brain can be used to detect pathological changes in patients with alzheimer's disease (ad). using time-resolved spectroscopy (trs) and phase-modulation spectroscopy (pms), we measured the absorption (ua) coefficient, scattering (us) coefficient, and mean photon pathlength (pl) of red light directed through the base of the frontal lobes of patients with ad and age-matched control subjects. the measured values and the asymmetry index (ai) (an indication of the symmetry of the measurements between the left and right side of the brain) were correlated with the severity of disease as determined by mini-mental state score. there were significant differences between the ad and control group for ua, us, pl, and the standard deviation of ai. there was no correlation between the mms score and ua, us, or pl. however, the highest asymmetry index values were seen in moderately impaired patients , which suggests that the asymmetrical nature of the pathological process detected by optical spectroscopy is most marked during this stage of the illness. this noninvasive technique may provide a convenient method to detect and monitor the pathological changes that occur in the brain of patients with ad. disease p . memory impairment in very mild alzheimer's a memory impairment is often the earliest indication of alzheimer's disease (ad). we investigated components of disease learning and recall to determine which aspect of memory function is impaired the earliest in incipient ad. using the mayo clinic alzheimer's disease patient registry, which is a longitudinal prospective project on ad and normal aging, we identified patients with very mild ad (i.e., with a mini-mental state score of or greater) and age-and sex-matched controls. we assessed performance on memory measures: the rey auditory verbal learning test and the buschke free and cued selective reminding test (fcsrt). the parameters evaluated included a measure of acquisition, total learning over trials (tl), and delayed recall (dr). on the fcsrt, an index of facilitation of performance with semantic cues (sc) was assessed. results indicated that all indices, tl., dr, and sc, were capable of separating the mild ad group from the controls ( p < , ). using a linear discriminant analysis with stepwise variable entry, the measure that assessed the patient's ability to use semantic cues (sc) was the most sensitive parameter for separating the groups ( f = . , p < . ), and the acquisition parameter (tl) was also useful at adding some additional predictive power (f = . , p < . ). the delayed recall measure, however, did not add anything to the previous measures. it appears that very early ad can be detected using appropriately structured memory tasks, and these procedures can be helpful in identifying at-risk individuals. alzheimer's disease (ad) has an insidious onset that is difficult to date reliably. we developed a standardized interview to provide objective criteria for dating the onset of different symptoms (memory complainr, performance problems, language deficits, disorientation, depression, behavior problems, and psychosis), yielding an estimated disease onset date. inrerrater reliability (icc = . ;p < , ) and interinformant reliability (icc = ; p < . ) for the onset of first symptom was high. interrater agreement for the order in which symptoms appeared was high (icc = . - . ) as was interinformant reliability for all symptoms except memory complaint. the interview was administered to patients with ad. mean estimate duration of illness was . years k . years and correlated significanrly with problems in instrumental activities of daily living. sixty-six percent had memory complaint and % had performance problems as their initial symptom. this technique provides a reliable characterization of disease onset. longitudinal studies will determine if particular onset symptoms differentially predict disease progression. the purpose of this study was to determine whether there is an excess of white matter disease (wmd) in alzheimer's disease (ad). brun and englund ( ) reported an excess of wmd in brains of patients with ad vs age-matched controls. there have been reports both confirming (bowen et al, ; fazekas et al, ) and refuting (leys, ) these findings using ct and mri in patients with clinically diagnosed ad. postmortem t -weighted mri scans and program and abstracts, american neurological association neuropathology were graded on brains of pathologically confirmed ad subjects and brains of age-matched neuropathologically normal controls. white matter lesions were scored on a to scale (none, mild, moderate, severe) separately for periventricular (pvl) and deep white matter (dwm) areas in mri scans and lux fast blue (lfb)-stained brain sections. correlations between mri and neuropathology were good ( r = . for pvl, r = . for dwm). pvl scores were higher in ad than in normal subjects on mri (ad: . l . vs controls: . rfr . ; p < . ). on pathology the difference in scores did not reach significance (ad: . . vs controls: . * . ; p = . ). similarly, dwm scores were higher in ad subjects than normals on mri, but not neuropathology. in conclusion, ad brains have a significant excess of wmd on mri compared to controls. although the pvl and dwm scores for pathological sections are not different in the groups, mri is much more sensitive than lfb-stained sections for wmd. thirteen autopsy cases of progressive supranuclear palsy (psp) were investigated for clinical-neuropathological correlations and heterogeneity. we reviewed clinical records of men and women aged to years (mean age yr) with disease duration ranging from to years. most patients had classic features of psp including ophthalmoplegia, postural instability, and extrapyramidal signs. dementia was eventually observed in of the patients ( %). six of patients ( %) on whom adequate initial documentation was available presented with memory loss or behavior change. five of the patients ( %), including with an initial presentation of memory loss, were diagnosed clinically as having alzheimer's disease (ad) rather than psp; neuropathological diagnoses in these cases varied: i had combined ad-psp; had ad-psp combined with parkinson's disease (pd) changes; had psp-pd; and had "pure" psp. the patients with concomitant pd changes showed lewy bodies in the substantia nigra, locus coeruleus, nucleus basalis, and neocortex. the remaining patients were clinically diagnosed as having psp; neuropathological diagnoses in these cases included with "pure" psp and with psp that also met neuropathological criteria for ad (psp-ad). these findings emphasize the clinical and neuropathological heterogeneity in psp. the neuropsychological battery developed for the consortium to establish a registry for alzheimer's disease (cerad) is currently used in many research studies to index the cognitive impairments of alzheimer's disease. in spite of its widespread use, normative informarion on the battery, important for interpretation of performance, has not been available. we report norms for the cerad battery based on a large sample of elderly control subjects (n = ; white men and women; ages - yr) enrolled in the national study of cerad. performance on the neuropsychological measures was examined separately for subjects with high ( yr) and low (< yr) education. distribution of scores and basic descriptive information (means and sd) for each measure were determined. significant age and sex effects were observed on most cognitive measures in the highly educated group. in contrast, no significant age effects were observed in the low education group. effect of sex was not explored in this group due to the limited sample size (n = ). further exploration of cerad performance in normal controls from underrepresented groups including minorities, residents of rural communities, and individuals with low education is in progress. intraneuronal inclusions of cytoskeletal proteins appear in several neurological diseases; for example, the neurofibrillary tangles of alzheimer's disease contain a cytoskeletal protein, tau. because the previously described slowing of axonal transport in aged animals might lead to accumulation of cytoskeletal proteins in nerve-cell bodies and axons, we assessed the abundance of major cytoskeletal proteins in brain tracts of rats at age months or months. immunoassay was performed with monoclonal antibodies to alpha and beta tubulin and to nf-l (the core neurofilament protein) by published methods. samples were dissected in a standardized fashion and -mrn pieces of the following tracts were assayed: optic nerve, corticospinal tract (medulla), superior cerebellar peduncle, l dorsal root, and l ventral root. between months and months, the nf-l content approximately doubled in each brain site. tubulin substantially increased at of aged rats all sites except the fimbria-fornix. in contrast, tubulin did not change in the spinal roots. nf-l increased slightly in the ventral but not the dorsal root. this tendency of senescent brain neurons to accumulate cytoskeletal proteins in their axoplasm may predispose them to formation of intraneuronal inclusions in various degenerative diseases. we performed a prospective study of preoperative magnetic resonance imaging (mri) in consecutive patients with intractable partial epilepsy who underwent a stereotactic resection of an extrahippocampal temporal lobe foreign-tissue lesion, "lesionectomy," between june and january . interpretation of the mri studies was performed by an investigator blinded to the presurgical evaluation, surgical outcome, and pathology. hippocampal formation (hf) atrophy was assessed using mri-based volumetry (n = ) and visual grading of the h f (n = ). mri-detected hf atrophy has been shown to be a reliable marker of moderate to severe mesial temporal sclerosis (mts) (cascino gd, et al, ann neurol ; : - evidence from experimental animals indicates that endogenously produced platelet-derived growth factor (pdgf) is an important regulator of glial proliferation and differentiation. because of the striking degree of glial proliferation in chronic epilepsy, we sought to determine whether cultured glia from human epilepsy tissue would be responsive to pdgf. the effects of pdgf on dna synthesis, proliferation, and relative distribution of a b (+) glia were studied in a cell culture derived from temporal lobe white matter of adult epilepsy lobectomy tissue. by immunocytochemistry, glial fibrillary acidic protein (gfap) was detectable in % of cells in untreated or -day pdgf-treated ( ng/ml) cultures, which confirmed their astrocytic nature. in contrast, a b ( +) cells increased from to % in untreated cultures to % after pdgf treatment, which suggested that type astrocytes (a b [ + , gfap[ +]) had been elicited. dna synthesis of cells resembling oligodendrocyte-type astrocyte ( - a) progenitors occurred within hours after program and abstracts, american neurological association pdgf treatment as evidenced by nuclear incorporation of brdu in bipolar a b ( +) cells. these studies demonstrate that expansion of adult human gfap( +) astrocyte populations is sensitive to regulation by pdgf. further, these data imply the existence of pdgf-responsive - a progenitor cells in astrocyte-rich cultures derived from human epilepsy tissue. ( we have recorded vagal and esophageal-evoked potentials after electrical (e) and balloon (b) stimulation in epileptic patients who had vagal stimulators for the control of intractable epilepsy and the results were compared with esophagealevoked potentials in healthy controls and diabetic patients. the vagal and esophageal-evoked potentials showed similar configurations with major positive and negative potentials. the amplitudes of the responses habituated rapidly over trials at per second up to per seconds. latencies were shorter from the upper esophagus ( cm above lower esophageal sphinctereles]) cf. lower esophagus ( cm above les) yielding conduction velocities of to meters per second but conduction was significantly slower in the diabetics. the vagal-evoked potentials have validated the use of esophageal-evoked potentials as a practical method of assessment of the integrity and speed of conduction in vagal afferent pathways in man. ronald e. kramer and neil l. rosenberg, englewood. co seizure disorders were analyzed in patients in whom toluene was the sole or major drug of abuse. toluene abuse is increasing; therefore, physicians should gain experience with its neuropathological and ciinical sequelae. a retrospective chart review found patients meeting criteria. the average patient age was years; abuse onset averaged . years; abuse averaged . years; and patients were male. ten were daily, were weekly, and were intermittent users. seizures occurred in . one suffered a single generalized tonic-clonic seizure without recurrence and without treatment. his we characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and - -methyldopa levels in patients with parkinson's disease (pd) and fluctuating responses to oral levodopa/carbidopa. dose response to graded intravenous levodopa was measured after overnight drug withdrawal on occasions, first after chronic, intermittent oral levodopa/ carbidopa and second after to days of continuous intravenous levodopa. continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, and reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/ - -methyldopa remained constant. our results support the hypothesis that relief of parkinsonism and production of dyskinesias occur by separate mechanisms. continuous dopamine-mimetic therapy should be sought as a therapeutic goal for advanced pd. dopa-responsive dystonia (drd) is a distinct subset of idiopathic dystonia with diurnal fl uctuation and a dramatically beneficial response to l-dopa. it has hitherto been considered an autosomal-dominant disease with reduced penetration (mckusick no. ) . we studied an arabic family of members with d r d spanning generations. we examined members and of their spouses. l-dopa was withheld for hours from patients in treatment. five family members had generalized dystonia with diurnal auctuation ( i male, in an arabic family females). dystonia started between the age of and years with gait difficulty and involvement of the legs. mri, eeg, evoked potentials, and screening for wilson's disease were negative. an excellent response to l-dopa was noted in all patients with continued long-term clinical stability for as long as years. the patients were the products of consanguineous marriages, and their siblings were normal. the patients were descendants of the same great-great-grandparents. this pedigree suggests an autosomal-recessive type of inheritance. we believe this is the first report of d r d with an autosomal-recessive type of inheritance. a. achiron, m . gornish, h . goldberg, i . ziv, r. djaldetti, y. zoldan, h. smka, and e. mekzmed, petah tiqva, israel freezing gait is an incapacitating symptom that occurs often in advanced parkinson's disease and also in other neurological disorders, eg., multiinfarct state, multisystem atrophies, and normotensive hydrocephalus. we evaluated, videotaped, and rated patients ( men, age k , - yr) who developed pure progressive freezing gait during . . , . - years. severity was mild in with sudden motor blocks mainly when confronted with obstacles; moderate in with gait arrests upon any attempt to initiate walking and changing direction, requiring a walking stick or partial external assistance; and severe in with total inability to start walking, requiring a walker, massive assistance, or a wheelchair. in all, freezing was associated with postural instability. they could mimic normal gait when seated or lying prone and could overcome arrests by the "walking over lines" maneuver. neurological examination was otherwise normal with no signs of dementia, parkinsonism, or pseudobulbar palsy. ischemic risk factors including ischemic heart disease, hypertension, and diabetes occurred in and previous strokes in . brain c t and mri were normal or showed mild cortical atrophy in and putative lacunae in only patients. none responded to levodopa or dopamine agonists. progressive pure freezing gait should be recognized as a separate nonparkinsonian neurological entity. it may be due to degenerative or ischemic non-nigral brainstem lesions. we describe patients with causalgia and dystonia, triggered by peripheral injuries in patients and occurring spontaneously in patients. the injury was often trivial. the mean age at presentation was . years. the legs were affected in patients, and the arm was affected in the remaining patients. all had burning pain, allodynia, and hyperpathia, along with vasomotor, sudomotor, and trophic changes. all developed typical dystonic muscle spasms in the affected part. the spasms typically were sustained, producing a fixed dystonic posture, in contrast to the mobile spasms characteristic of idiopathic torsion dystonia. dystonia always followed the causalgia and was painful. there was spread of the causalgia and of the dystonia from its initial site both in the affected limb and to other extremities, the latter in a hemiplegic, transverse, and triplegic distribution. all forms of conventional treatment failed to relieve either the pain or the dystonia. we suggest that functional changes in the corticobasal ganglia-thalamic system are responsible for this painful dystonic syndrome. program and abstracts, american neurological association holiday for parkinson's disease: a controlled clinical trial r. kurlan, c . m . tanner, c. g. goetz, j . sutton, p. carvey, c. deeley, l. cui, c. itvine, and m . mcdemzott, rochester, ny, chicago, il, and san jose, c a the efficacy and mechanisms of levodopa (ld) drug holiday for parkinson's disease (pd) remain controversial. we performed a double-blind, randomized study with advanced pd patients ( men, women; aged - yr) with entry criteria of inadequate response to ld plus dose-limiting ld-induced side effects (dyskinesias, hallucinations, and confusion). subjects were assigned to: ( ) % placebo for ld (complete drug holiday) or ( ) % ld and % placebo for ld ( % drug reduction) for days. after subsequent open-label ld dose optimization, subjects were followed to end point (defined as the time when entry criteria were again satisfied or a maximum of year). median survival time to end point was not significantly different for the complete drug holiday ( days) and % drug reduction ( days) groups ( p = ) . aspiration pneumonia occurred in complete drug holiday patients and no significant morbidity occurred with drug reduction. after a -mg dose of ld, clinical and pharmacological responses were no different before and after drug holiday ( p = . ) or reduction ( p = ). subject to the limitations of our small sample size, we conclude that complete drug holiday is associated with greater morbidity and confers no major advantage over % drug reduction. we found no evidence of significant alterations of pharmacokinetic or pharmacodynamic properties of ld after drug holiday or reduction. ( (pc) of the substantia nigra on t -weighted images. the narrowing of the pc signal has been attributed either to atrophy of the pc or to increased deposition of iron in this region. we have studied details of iron distribution in the midbrain of formalin-fixed human brains by scanning pixe analysis. three p d brains, juvenile pd brain, and control (amyotrophic lateral sclerosis) brains were studied. in the controls, the iron content of the pars reticulata (pr) was almost equal to that of the red nucleus (rn), but that of the pc was less than % of the pr. in parkinsonian brains, the iron content in the pc was significantly higher than in the controls. the iron content ratios of pc/pr and pc/rn in parkinsonian brains were significantly higher than in the controls. these findings suggest that iron deposition increases in the pc of parkinsonian brains. the difference in the pattern of iron content corresponds to the intensity profile pattern noted on mri. mri findings in parkinsonian patients may reflect a change in the iron distribution pattern. (jankovic and brin, nejm, ) . such resistance within exposures is not likely due to toxin antibody formation. of cd patients evaluated per protocol receiving or more botox tx under multichannel emg monitoring, ( . %) showed no benefit after the first and second tx, despite mild neck weakness on static muscle testing and, in some instances, emg signs of denervation. the responders ( men, women) had younger age onset cd (mean yr vs yr), but similar duration (mean yr vs yr) compared to the male and female nonresponders (in contrast to jankovic and schwartz, arch neurol, ) . both groups had similar degrees of severity and tx dose (mean iu, range . - ). although disparate group size prohibits statistical analysis, some interesting comparisons include: nonresponders were more apt to have extranuchal dystonic sites ( % vs %), antecollis ( % vs %), dark eyes ( % vs %), women with elevated antinuclear antibody titer ( % vs %), history of intracranial operation ( % vs o%), significant for age focal mri abnormality ( of vs of ). history of cervical operation ( patients) did not limit responsiveness. rates of prior remission, perinatal stress, antecedent trauma, left-handedness, and family history of movement disorder were similar for both groups. antecollis presents problems for optimizing tx to affected muscles, but central mechanisms may play a role in why some patients with focal dystonia do not improve with botox tx from the outset. enrico fazzini, new york, n y with parkinson's disease does deprenyl have a symptomatic effect on patients with untreated and l-dopa-treated parkinson's disease (pd)? once deprenyl is started, how long is it before another medication is needed to control symptoms of continued disease progression? there has been controversy over whether deprenyl has effects on delaying disease progression (nejm ; : ) and/or in alleviating the symptoms of pd. one hundred seventy-five patients already taking l-dopa (group ) and patients who had never taken l-dopa (group ) were treated with deprenyl mg/day. unified pd rating scale (updrs) scores were measured before and after deprenyl. patients were followed until pd symptoms progressed to the point of requiring additional medication. one hundred eighteen of ( %) patients in group (reduced updrs mean activities of daily living [adl) to , motor [mtr] to ) and / ( %) patients in group (reduced updrs mean adl to , mtr to ) reported symptomatic benefit. an average of months' duration was found in both groups before further medication adjustments were needed. deprenyl provides symptomatic benefit for an average of months in the majority of patients with p d regardless of whether or not they are being treated with l-dopa. yasuo iwasaki, masao kinoshita, toshiya shojima, and ken ikeda, tokyo, japan, and cleveland, oh in parkinsonian patients we measured fasting plasma amino acids in parkinson's disease patients and controls matched for age and sex. all patients were receiving l-dopa and they were free of any medications other than l-dopa. normal controls were free of any medication. there were no differences in diets between patients and controls. fasting blood specimens were collected in heparinized tubes and immediately were centrifuged at , g for minutes. analysis of plasma amino acids was performed by automated ion-exchange chromatography with lithium-based buffer and an amino-acid analyzer. parkinsonian patients had significant elevations of aspartate, glutamate, and glycine. the other amino acids were not significantly different from those in controls. n o correlation between severity or activity and degree of abnormality in plasma level of amino acids in patients was established. we conclude that excitatory amino-acid metabolism is altered in patients with parkinson's disease. bonnie e. levin, rachel tomer, and william weiner, miami, fl disease there is evidence linking obsessive-compulsive symptoms (ocs) to basal ganglia dysfunction. we investigated the presence and severity of ocs in a sample of patients with an unequivocal diagnosis of idiopathic parkinson's disease (pd) using the leyton obsessional inventory. ocs was found in the majority of the patients, with ( %) scoring above the normative cutoff for the symptom score and ( %) scoring above the normative cutoff for the trait score. when severity of oc symptoms was correlated with a battery of neuropsychological measures, significant relationships were observed between ocs and a preponderance of tests associated with right-hemisphere functions. these findings were observed especially on those tests with a strong frontal lobe component (block design: r = -. ; embedded figures: r = -. ; set shifting: r = -. ; and perseverative responses: r = . ; p < . for all measures). in all cases, the more severe oc symptoms, the poorer the performance. a similar trend was observed between the leyton trait scores and the cognitive measures. these findings suggest that ocs is present in a subgroup of pd patients, which may reflect greater compromise of right-hemisphere basal ganglia-frontal lobe pathways. intraclass correlations (iccs) were calculated for the total motor score and for each individual sign. results indicated excellent agreement (icc > . ) for the total motor score, resting tremor, gait, arising from a chair, and speeded, repetitive movements; good agreement (icc > . ) for rigidity, action tremor, posture, postural stability, and bradykinesia; and poor agreement (icc < . ) for speech and facial mobility. a factor analysis was then performed on updrs motor scores for pd patients from a community-dwelling cohort. three factors were extracted by principal components analysis with subsequent varimax rotation, accounting for . % of the total variance: factor -balance and stability (posture, postural stability, gait, arising from a chair, and bradykinesia); factor -rigidity and motor speed (rigidity, speech, facial mobility, rapid alternating movements, leg agility, hand movements, and finger tapping); factor -tremor (resting and action). these results indicate that the updrs motor examination is reliable between raters and measures the cardinal signs of pd. an open pilot study was performed to evaluate the efficacy of botulinum a toxin (botox) injections for disabling hand tremors. a previous report on the use of botox for hand tremors suggested that it was helpful, but relied on subjective clinical rating scales. the extent of normal clinical fluctuations or a placebo response could not be determined. to investigate these issues more objectively, patients with parkinson's disease and with essential tremor with refractory hand tremors underwent electromyographically guided intramuscular injections of botox into wrist flexors and extensors. patients without great medication-related tremor fluctuations were selected. results before and after botox were determined by comparing ( ) patient perceptions of functional improvement, ( ) clinical assessments using the unified pd rating scale for tremor and the webster rating scales, and ( ) physiological measurements using accelerometric analysis of hand tremors of tremor frequency, amplitude, and waveform characteristics. all patients reported some improvement, ranging from mild to marked with a mean of . on a to ( = marked) global rating scale. however, only / patients showed a significant improvement in the clinical rating scales, confirmed by > % reduction in tremor amplitudes. these findings show that most patients reported improvement not confirmed by the clinical or physiological measures. efficacy of botox injections for tremors is implied, but controlled trials are needed before this procedure can be generally recommended. christopher g. goetz and glenn t . stebbins, chicago, i l we tested whether hallucinations, motor disability, and cognitive decline were risk factors for nursing home placement in advanced parkinson's disease (pd) and whether these effects were independent or synergistic. between and , we identified patients admitted to long-term nursing homes. using case control methodology, we matched each for age, pd duration, and sex with control pd patients remaining at home. parkinsonism was assessed by the motor and activities of daily living subscales of the unified p d rating scale (updrs); hallucinations and dementia were determined by scores on the thought disorder and intellectual impairment items of the updrs. tests of synergy were based on a mantel-hentel model. hallucinations were a significant risk factor with odds ratio = . , x = . , p < , . motor impairment alone and cognitive impairment alone were not significant risk factors for nursing home placement (x for motor severity = , , p > . , and x for cognitive impairment = . , p > . ). furthermore, combined odds ratios for hallucinationslmotor severity and hallucinations/cognitive impairment showed no synergy of effect (x < . for both,p > . ). of the variables studied, hallucinatory behavior is the most prominent and independent risk factor for nursing home placement in these patients; the data suggest that aggressive control of hallucinations may be warranted to prevent nursing home admission. temperature-sensitive paramyotonia congenita phenotype* louis j . ptacek, philip mcmanis, hzrbert kwiecinski, alfred george, robert barchi, launce gouw. and mark leppert, salt lake city, u t , rochester, mn, warsaw, poland, and philadelphia, pa the periodic paralyses are a group of autosomal-dominant muscle diseases sharing a common feature of episodic paralysis. in one form, paramyotonia congenita (pc), the paralysis is temperature-sensitive, usually occurring with muscle cooling. electrophysiological studies of muscle from patients with pc have revealed temperature-dependent alterations in sodium channel (nach) function. this observation led to the identification of distinct mutations in an s segment of a skeletal muscle nach in unrelated pc families. we describe the use of the single-strand conformation polymorphism (sscp) technique to define a third allele specific to pc patients in an additional family. this aberrant pattern, though distinct from the first , occurs in the same exon of this nach gene. sequencing is currently underway to define the molecular alteration causing this aberrant pattern. two additional families with the pc phenotype have been sampled and do not demonstrate these sscp variants. we are currently searching for new mutations in these families to define further the molecular heterogeneity of this temperature-sensitive pc phenotype. parag mehta and roger w. kukz, brooklyn, n y abnormal accumulation of calcium (ca) in myofibers is thought to play a role in pathogenic myonecrosis. attempts at reducing intracellular ca content with ca channel blockers in duchenne muscular dystrophy (dmd) have been clinically unsuccessful. dantrolene, however, which acts at the sarcoplasmic reticulum to inhibit ca release from intracellular stores, has produced dramatic reductions in serum creatine kinase (ck) in dystrophic mice and more recently in dmd. we investigated the effect of low-dose dantrolene in a group of patients with limb girdle dystrophy (lgd), dmd, and other myopathic disorders. all subjects received dantrolene in incrementing doses from to mg daily over a to -week period. mean baseline ck was compared to ck with dantrolene treatment. dramatic reductions in serum ck levels averaging % were seen at to -mg doses in lgd patients ( ). dmd patients ( ) and patients with other myopathies ( ) showed a similar but less dramatic reduction in ck. three of the weakest patients complained of increased fatigue while taking mg, which suggests that higher-dose dantrolene may confound longer-term trials assessing clinical muscle strength and function. dantrolene in dosages well below conventional antispastic doses has a dramatic effect on serum ck and possibly myofiber necrosis in lgd, other dystrophies, and other muscle disorders. p . antigen-specific therapy in myasthenia gravis: myasthenia gravis (mg) is mediated by anti-acetylcholine receptor (achr) antibodies, believed to be t-cell dependent, and antigen-specific therapy would be preferable to current nonspecific immunosuppression. exposing mouse t-cell clones to mhc class i molecules complexed with relevant antigen on planar membranes induced proliferative unresponsiveness (quill and schwartz, ) , and soluble mhc class i molecules complexed with myelin basic protein (mbp) peptide resulted in unresponsiveness of specific tlymphocyte clones in vitro (sharma et al, ) . we have used our well-defined dr -restricted t-cell clone (ong et a [, ) isolated from an mg patient and specific for p - of the achr alpha subunit. overnight incubation of these t cells with a soluble p - : dr complex substantially inhibited the subsequent response to challenge with soluble antigen and presenting cells. in contrast, antigen response after preincubation with dr complexed to an irrelevant peptide (mbp - ), soluble dr alone, or an experimental approach studied in vitro p - alone (at equimolar concentrations) did not differ appreciably from that in untreated cells. the p - :dr complex had no effect on other non-achrspecific cell lines/clones. these results suggest that the use of soluble mhc-peptide complexes may be an approach to selective immunotherapy in mg patients. p . high-dose intravenous immunoglobulin in the shawke a. soueidan and marinos c. dalakas, bethesda, md inclusion body myositis (ibm) is a severe disabling inflammatory myopathy with characteristic clinical and histological features. it is commonly suspected when a patient with presumed polymyositis does not respond to available immunotherapies. the need for an effective treatment in patients with ibm prompted the present pilot study using high-dose intravenous immunoglobulin (hd-ivig), an apparently effective immunomodulating agent in several autoimmune neuromuscular disorders. we treated patients with muscle biopsy-proven ibm with up to monthly infusions of gml kg ivig. after the first infusion, of the patients showed definite functional improvement consisting of independent ambulation, fewer falls, and increased ability to lift weights. the muscle strength of the proximal and less atrophic muscle groups improved by one grade mrc scale (from to ), whereas the distal and atrophic muscles remained unchanged. the improvement, sustained up to months, was greater in patients with the most severe endomysial inflammation. we conclude that hd-ivig may be the first promising agent that can improve the strength of certain muscle groups in patients with ibm. because ivig is prohibitively expensive, the present encouraging results warrant a large-scale controlled therapeutic study. hays, and n . lutov, new york, n y , and milan, italy anti-myelin-associated glycoprotein (mag) antibodies from patients with neuropathy cross-react with the glycolipid -sulfated glucuronyl paragloboside (sgpg). among patients tested by enzyme-linked immunosorbent assay and western blot, had highly elevated antibody titers ( , ) to both mag and sgpg, had highly elevated titers to mag alone, and had highly elevated titers to only sgpg. immunostaining of normal nerve myelin by the antibodies correlated better with anti-mag than anti-sgpg activity. twenty-one of the patients, including patients in all groups, had predominantly sensory or sensorimotor neuropathy, and biopsy specimens revealed deposits of igm and complement on affected myelin sheaths. three patients presented with motor syndromes, all with antibodies specific for sgpg; had a predominantly motor demyelinating neuropathy, had upper and lower motor neuron signs and peripheral neuropathy, and had amyotrophic lateral sclerosis confirmed post mortem. all had deposits of complement on peripheral nerve myelin sheaths. these studies suggest the following: ( ) that anti-mag or sgpg antibodies may differ in their fine specificities and biological activities, ( ) that anti-sgpg antibodies also may occur in motor neuron diseases, complicating the clinical presentation, and ( ) that both mag and sgpg should be used as antigens in testing for autoantibody activity in peripheral neuropathy. david b. williams, john steele, ulla-katrina craig, sandra bryant, peter o'brien, and leonard kurland, newcastle, new south wales, australia, mangilao, guam, and rochester, m n continuing surveillance of neurodegenerative diseases in the mariana islands reveals changes in frequency and clinical characteristics since the s that resemble those in other known western pacific foci (kii peninsula, japan, and irian jaya, new guinea). recent surveys of patients years and older were conducted on rota, tinian, and yigo, guam. possible cases of dementia, parkinsonism, and amyotrophic lateral sclerosis (als) were identified by local trained personnel using a questionnaire, world health organization neurology test, and cognitive screening. those who failed the screening were examined by a neurologist. in the small populations of rota and tinian, there were no definite cases of als compared to i to cases present in previous surveys. the high prevalence of parkinsonism-dementia complex (pdc) was unchanged and dementia was increased compared to earlier surveys. in yigo, als and pdc continue to be prevalent; however, the als patients are predominantly long-term survivors (> -year disease duration). in areas of previous high prevalence of als/pdc, dementia (as pdc) was associated with extrapyramidal signs, whereas in areas of previously low prevalence of als/pdc, dementia alone, possibly of alzheimer type, predominated. these observations help to confirm previous reports of changing clinical patterns, but suggest that the geographic distribution of the (presumed) environmental etiological agent for als/pdc remains stable after almost years. p. v . fragokz, . frongillo, m. michisanti, g. antonini. derangements of the cardiac conducting system are the most common features of heart involvement in myotonic dystrophy (md). in view of chis patients with various grades of md ( males and females, mean age yr) underwent -lead ecg and holter monitoring. in patients ( %), almost all with a severe grade of md, or more conduction defects were found: first-degree atrioventricular block (i-avb) in cases, second-degree avb in case, right bundle branch block in cases, left anterior hemiblock in cases, left bundle branch block in cases, and trifascicular block in case (pacemaker implanted). an -year-old boy had a chronic atrial fibrillation with slow ventricular rate; he died suddenly while awaiting electrophysiological study. thirty-seven patients were followed over a mean period of months (range - mo). a -year-old woman experienced a myocardial infarction and was excluded from subsequent considerations. conduction defects de novo appeared in patients: i-avb in and i-avb plus -avb (mobitz i and i type) in . nine patients, all with i-avb at initial evaluation, showed deterioration of their defects' conduction: a bifascicular block was observed in cases and a trifascicular block in cases (pacemaker implanted). conduction defects may run a malignant course in md, mainly in patients with more severe grades of the neuromuscular disease; thus, a close cardiological evaluation is mandatory for a proper therapeutic approach in single cases. hiroshi mitsumoto, surest kumar, kevy h. levin, robert w. shields, jr, michelle secic, asa j . wilbourn, and rajendra g . desai, cleveland, oh, and santa ana, ca twenty patients with amyotrophic lateral sclerosis (als) entered a pretreatment study with monthly quantitative isometric muscle strength tests ( muscles in each extremity) and quantitative tufts scales including vital capacity, bulbar diadochokinetic rate, timed water drinking, timed rising from a chair, and timed walking meters. after to months of pretreatment observation, the patients received mg/kg intravenous immunoglobulin (ivig) (gamimune-n, miles) every month for up to months. during the study period, patients died and patients withdrew from the study. the slope of each variable's changes over time before and after the ivig treatment were compared statistically. none of the quantitative scales showed significant change with ivig. however, the slope of the upper extremity muscle strength revealed improvement with ivig treatment ( p = . and . , right and left, respectively). when all extremities were combined, the slope was also significantly improved with ivig ( p = . ). lower extremity muscle strength alone showed similar trends but no statistical significance. electrophysiological and immunological data were also analyzed. our results warrant a double-blind, controlled study with ivig for the treatment of als. leber's hereditary optic neuropathy (lhon) is a mitochondrial disorder with predominantly optic nerve abnormality. it can be associated with dystonia, ataxia, encephalopathy, cardiac abnormalities, and other less well-characterized neurological syndromes. we describe members of a family with lhon with a slowly progressive motor polyneuropathy. a -year-old man and his -year-old sister have had mild motor impairment since early childhood. a gait disorder and distal muscle weakness became evident at puberty. the girl, but not the man, also has blindness, distal numbness, type i diabetes mellitus, and short stature. physical examination showed in both: bilateral foot drop, limb hyperreflexia but absent ankle reflexes, distal sensory loss, and a slight brownish scaly skin discoloration over the forearms. only the girl had clonus and optic nerve atrophy. the man had peripapillary telangiectasias. the jaw jerk was normal in both. motor nerve conductions in both showed absent tibia and peroneal responses, whereas other motor and sensory nerve conductions were normal. emg revealed denervation, more so distally. muscle biopsy findings showed recent denervation and previous denervation followed by reinnervation. n o raggedred fibers were observed with the modified trichrome and sdh stains. brain mri was normal in both. cerebrospinal fluid in the girl was normal. blood samples from both patients and maternally related family members revealed a mitochondrial dna point mutation at position (d. c. wallace). in this family, only male had lhon; females had lhon and others, including the patients' mother, were asymptomatic carriers. this association of chronic motor neuropathy and hyperreflexia with lhon appears to be a distinct syndrome. the pathogenic mechanism by which the mitochondrial dna defect causes the neuropathy (and other neurological deficits) requires analysis. duchennelbecker muscular dystrophy henry j . kaminski, mazen al-hakim, r. john leigh, bashar katirji, and robert l. ruff; cleveland, oh fast-twitch extremity muscle fibers are preferentially affected in duchenne/becker muscular dystrophy (dbmd). since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased among these patients. to investigate involvement of extraocular muscle (eom) by dbmd, we studied with infrared oculography patients who were wheelchair-bound and able to perform only minimal activities of daily living. saccades were slightly slowed but were within % confidence limits of normal. all patients showed square wave jerk movements (swj). in patients, the frequency of the swj exceeded that of normal subjects, which suggested central nervous system dysfunction. clinical neuroophrhalmological examination of other dbmd patients was normal. this investigation is the first study of ocular motility in dbmd and demonstrates that eom function is relatively preserved even in far advanced patients. eom is composed of a heterogenous mix of fiber types that differ in anatomical and physiological characteristics from extremity muscle. study of eom in dbmd may prove to be useful in understanding why some muscles are resistant to dbmd and in characterizing properties that limit muscle degeneration. (supported by nih grants ey , ey , the department of veterans affairs, and the evenor armington fund.) since patients with myotonic dystrophy (mtd) exhibit a marked resistance to insulin effect on glucose uptake and an impaired handling of the insulin-sensitive amino acids, it is possible that muscle wasting in mtd may reflect a derangement of insulin action on muscle protein metabolism. increased muscle protein breakdown in mtd would be expected if the normal inhibitory effect of insulin on protein catabolism is impaired. the forearm perfusion technique combined with measurements of -methylhistidine ( -mh) arteriovenous (a-v) differences by high-performance liquid chromatography provides a unique method to investigate skeletal muscle myofibrillar protein degradation in vivo. we studied -mh (a-v) and efflux from the forearm muscles in men moderately affected with mtd and normal men. efflux values (q) were calculated as the product of -mh (a-v) times forearm plasma flow measured by the indicator dilution technique. forearm -mh release (estimated as {a-v} or q) of mtd patients did not differ significantly from normal controls. we conclude that myofibrillar degradation is not increased in mtd even when measured in a muscle compartment selectively affected by wasting. the possibility of an impaired anabolic action of insulin in mtd has yet to be determined. to study the relationship between the ragged red fibers (rrf) and age, we have reviewed muscle biopsy specimens prospectively. patients with well-established mitochondrial myopathy syndrome and with myopathies known to produce secondary rrf were excluded. the number of ragged red fibers (rrf) was counted under x (lpf) magnification. rrf were identified by the modified trichrome and sdh stain. for the final analysis, the sdh staining was used. the frequency of rrf was analyzed in relation to patients' ages. the frequency of cases with more than rrf increased with aging: % in the first decade, % in the fourth decade, and % in the eighth decade. the frequency of cases with more than rrf also increased with aging: % in the first three decades, % in the fourth decade, and % in the eighth decade. of patients with well-established mitochondrial myopathy syndrome, had more than rrf under lpf. the number of rrf in muscle increases with aging, indicating that mitochondrial activity in muscle is affected by aging. this finding may complicate the diagnostic criteria of mitochondrial myopathy in older individuals. ten rrf under lpf seems to be a reasonable cutoff point for the diagnosis of mitochondrial myopathy. schwartz-jampel, a rare autosomal-recessive syndrome characterized by short stature, myotonia, skeletal abnormalities, and peculiar facies, was reported by aberfeld in . the same sibship was earlier reported by schwartz and jampel in with emphasis on blepharophimosis. as of this writing about cases have been reported in the literature. most of the features of this syndrome are believed to be secondary to primary muscle disease. several peripheral electrophysiological studies showing features of myotonia have been reported. we describe patients with schwartz-jampel syndrome showing evidence of central conduction disturbance documented by somatosensory-evoked potentials (seps). median nerve seps showed normal latencies to erb's point and n- in all. interpeak latencies between n and n were prolonged in with complete block in . emg showed typical myotonic discharges in all. motor nerve conduction velocities, visual and brainstem auditory-evoked potentials, ct, and mri were normal in all. seps in the parents were normal. we believe this is the first report documenting evidence of central nervous system (cns) involvement in schwartz-jampel syndrome. schwartz-jampel syndrome and myotonic dystrophy may have similar cns ''lesion'' as sep abnormalities also have been shown in myotonic dystrophy. epidemiological studies have associated consumption of certain batches of l-tryptophan (lt) with development of the eosinophilia-myalgia syndrome (ems). , '-ethyledenebisltryptophan) (ebt or peak e), a derivative of lt, is a trace contaminant associated with implicated batches of lt. three female lewis rats received ebt, mg per gm daily, by intraperitoneal injection. four control rats received unimplicated lt. n o peripheral eosinophilia, rash, or weakness were observed in either group. one rat from each group died during the experiment (control-bowel infarct; ebt-death under anesthetic). after days, forelimb and hindlimb muscles of the remaining animals were frozen and fixed for program and abstracts, american neurological association ultrastructural and histological studies. two ebt rats had a myopathy involving soleus with a perimysial infiltrate containing lymphocytes, macrophages and sparse eosinophils, and necrotic fibers; the other showed few necrotic fibers in gastrocnemius. occasional eosinophils were seen in fascia in both animals given ebt but not in controls. fiber-type specific quantitative analysis of the microvasculature showed no decrease in the capillary index. ultrastructural examination revealed an increase in the size of microvessels in ebt animals. no denervation or reinnervation were demonstrated. the perimysial inflammation replicates an important feature of human ems and supports the epidemiological evidence that ebt is the causative agent of the disease. britta ostermeyer-shoaib, bernard m. patten, and tetsuo ashizawa, houston, t x five women (patients - ) developed motor neuron disease (mnd) years (range - yr) after receiving silicone gel-filled breast implants. at explant in patients, had both and had the left implant ruptured with silicone spilled into tissue. one woman (patient ) developed amyotrophic lateral sclerosis (als) years after numerous injections of free silicone into her face. biceps muscle biopsy specimens in all showed neurogenic atrophy. patient developed als with bulbar involvement and died years later of respiratory failure. she had anti-gm antibodies and autopsy findings confirmed the diagnosis of typical als. patient developed als, but also fatigue, myalgia, arthralgia, and skin rash. she had anti-gm antibodies, antisilicone antibodies, positive antinuclear antibodies (ana), and decreased serum igg, iga, and c , but increased igm and creatine phosphokinase (cpk) and chronic inflammation was revealed in muscle biopsy specimens. patient developed als, but also had hair loss, skin rash, fatigue, headache, sjogren's syndrome, and positive ana. patient developed als, but also had myalgia and arthralgia. patient developed lower mnd, but also fevers, arthralgia, and joint stiffness. she had anti-gm antibodies, positive ana, antimyelin antibodies, and decreased serum igg and iga with chronic inflammation shown in nerve biopsy findings. patient developed a steroidresponsive and steroid-dependent als with bulbar involvement. she had a monoclonal gammopathy in the cerebrospinal fluid and increased cpk. we suggest that silicone acts as an adjuvant that damages motor neurons via an indirect autoimmune mechanism. implants and silicone injections into the face p . silicone adjuvant breast disease: more forty-five women developed mixed sensory-motor neuropathy ( ), motor neuron disease ( ), multiple sclerosis ( ), multiple sclerosis-like syndrome ( ), or myasthenia gravis ( ) years (range mo- yr) after receiving silicone-gel breast implants ( ), saline-filled silicone-covered breast implants ( ), or direct injections of silicone into the breast ( ). most patients had, in addition, severe fatigability, myalgia, arthralgia, morning stiffness, skin rash, lymphadenopathy, sjogren's syndrome, and short-term memory problems. laboratory results revealed in most of the women decreased or increased serum immunoglobulins, autoantibodies, a serum monoclonal gammopathy, or oligoclonal bands in cerebrospinal fluid. at explantation in , had both and had implant neurological cases ruptured. biopsy of the fibrous implant capsule in most patients showed foreign-body giant cells containing refractile material consistent with silicone whether or not the elastomer shell was ruptured, indicating silicone bleed. the major finding on surd nerve biopsy was loss of myelinated fibers, on biceps muscle biopsy was neurogenic atrophy, and on pectoralis muscle biopsy was myositis with vasculitis and free silicone in some. we suggest that silicone may provoke damage to nerve and muscle, probably indirectly promoting autoimmunity. james f. howard, jr, m . kathleen donovan. and m. susan tucker, chapel hill, nc urinary symptoms of urgency and incontinence have been reported only rarely in patients with myasthenia gravis (mg) and then most often in association with myasthenic crisis. we report the case of a -year-old woman who in december had the onset of chest pain and was found to have a lymphocytic thymoma. in june she developed urinary incontinence, was found to have an open bladder neck. and underwent a suspension procedure for stress incontinence in january . eight months later she developed exertional fatigue and a diagnosis of m g was made. in july there was a recurrence of urinary incontinence. these symptoms clustered toward the end of the day and at trough mestinon dose. neuro-urophysiological studies demonstrated her previous open bladder neck, the inability to sustain a pelvic floor contraction, and increased bladder wall contraction. singlefiber electromyography (sfemg) recordings from the anal sphincter demonstrated a mean consecutive difference (mcd) of ysec, and % of fiber pairs had impulse blocking while recordings in the extensor digitorium communis muscle were normal. following a course of plasma exchange, there was significant clinical improvement with a reduction in the frequency of urinary incontinence, and improvement in anal sphincter sfemg studies (mcd, ysec with no blocking). this case demonstrates that in those myasthenic patients with predisposing bladder outlet dysfunction, urinary incontinence may be a manifestation of worsening mg. diana m. escolar, mohamed eldaly, and jaime rich, boston, m a debate still exists as to the role of antibody versus celmediated factors in the pathogenesis of guillain-barre syndrome (gbs). we describe a patient with increased proportion of circulating t cells and a t-cell lymphoma who developed gbs and responded to intravenous immunoglobulin (ivig). a -year-old man with t-cell lymphoma drveloped gbs by clinical, nerve conduction, and cerebrospinal fluid criteria. he had an elevated proportion of t cells and markedly reduced b cells with a normal cd /cd ratio. he responded rapidly to ivig, with return of nearly normal motor function in week. three weeks later, he relapsed and his vital capacity dropped. ivig was again administered and within hours he nearly recovered. a third relapse, days later, again responded to ivig. he has remained asymptomatic with ivig maintenance. the role of t-cell lymphocytes in initiating experimental autoimmune neuritis has been shown by adoptive transfer experiments. this patient with t-cell neoplasia may represent an analogous model in hu-guillain-barre syndrome mans supporting the role of t-cell (cell-mediated) autoimmunity in the pathogenesis of gbs. ivig therapy may act primarily by inhibiting the t-cell-mediated attack on myelin. p . sympathetic skin response: age effect it is frequently stated that the sympathetic skin response (ssr) can be elicited in all normal subjects, but the age of the investigated population usually is not considered to be a significant factor. we have examined the ssr in the upper and lower limbs of normal subjects, aged to years ( of them males). the ssr was elicitable in the lower limbs in all subjects under the age of years and in the upper limbs in all subjects younger than years. in contrast, it could be elicited in the lower limbs in only % and in the upper limbs in % of octogenarians. the amplitude of the response, though highly variable, showed a remarkable decline with age, both in the upper ( p < . , r = . ) and in the lower ( p < . , r = . ) limbs. these results indicate that age affects both the elicitability and the amplitude of the ssr. this has to be taken into consideration when evaluating the autonomic function in the elderly. we reviewed records of patients appearing to have motor neuron disease (mnd) to whom we recommended immunosuppression over years ( of m n d patients). atypical findings engendered hope rhat they might have treatable neuropathy. electrophysiological studies were mainly consistent with mnd, but also showed conduction block or other evidence of relatively mild peripheral nerve disease in . sensory symptoms were present in ; or more reduced or absent deep tendon reflexes in ; elevated cerebrospinal fluid protein in ; and nonspecific abnormalities on sural nerve biopsies in of . anti-gm levels were measured in (including who improved), but none was significantly elevated. immunosuppression included cyclophosphamide ( patients); prednisone ( ); plasma exchange ( ); intravenous gammaglobulin ( ); cyclosporine ( ); and total lymphoid irradiation ( ). seven treated patients died, worsened, remained stable for years, and improved. two patients declined treatment. one died and the other did not worsen in years. we conclude that immunosuppression by our methods is, at best, rarely effective in atypical mnd. we studied serum antiglycolipid antibodies by enzymelinked immunosorbent assay in patients with typical miller fisher syndrome (mfs), patients with atypical mfs who were lacking in some of the cardinal signs, patients with guillain-barrc syndrome (gbs) with ophthalmoplegia, patients with gbs without ophthalmoplegia, patients with multiple sclerosis (ms), and patients with other immunological disorders (oid) including systemic lupus erythematosus, polymyositis, and mixed connective tissue disorder. all patients with typical mfs had increased activity of igg antibody against ganglioside g q l b in the early phase, and it reduced with time. such anti-gqlb igg activity also was detected in of the patients with atypical mfs and in of the patients with gbs with ophthalmoplegia. in atypical mfs, the only patient without increased anti-gq b igg activity demonstrated normal eye movement with ptosis, whereas eye movement was impaired in the other patients. no patients with gbs without ophthalmoplegia, ms, or oid had increased anti-gqlb igg activity. these findings suggest the close association between increased anti-gql b igg activity and impaired eye movement in mfs and gbs. serum anti-gqlb igg activity possibly plays a role in impaired eye movement in mfs. our goal is to develop an assay that can be used to monitor a relevant immune effect of interferon p (ifnp) in multiple sclerosis (ms) patients during the course of ifnp immuno-' therapy, since recombinant ifnp is being tested in multicenter clinical trials. this report extends our prior studies of the inhibitory effect of ifnp on t-cell activation. peripheral blood mononuclear cells (pbls) from healthy donors and clinically stable ms patients were studied. pbl cultures were stimulated with cona, mab to cd , or with the phorbol ester pma in the presence of the calcium ionophore ionomycin. parallel cultures were studied in the presence of ifnp, uiml. t-cell activation was monitored by determining the percent cells positive for il- receptor (il- r) using facs analysis, or with a sensitive enzyme-linked immunosorbent assay for ifny. ifnp markedly inhibited il- r expression induced by cona, by mab to cd , or by pma and ionomycin, which activate t cells via different pathways. the results suggest that ifnp inhibits t-cell activation by actions independent of membrane receptors. we observed significant inhibition of cona-induced t-cell il- r expression in both ms patients ( . % inhibition, p < . ) and controls ( . % inhibition, p < . ). there was no significant difference in percent inhibition between ms and controls, but there was more variance among the ms patients. variability of biological effects of ifnp on t cells may relate to differential therapeutic responses to exogenously administered ifnp in ms patients. preliminary experiments suggested that ifnp inhibited ifn gamma secretion by pbl stimulated with cona. ifnp inhibits a number of events associated with t-cell activation, in both normal and ms t cells. response to ifnp appears more variable in the ms cases. immunological monitoring of t-cell activation in patients receiving ifnp may assist in understanding the observed therapeutic responses and planning clinical protocols. myelin destruction is associated with many central nervous system disorders, such as multiple sclerosis, head trauma, and ischemic injury. inflammatory cells, monocytes/macrophages, and polymorphonuclear leukocytes (pmn) may me-program and abstracts, american neurological association diate myelin injury, and lipid peroxidation may be an important mechanism. inhibiting myelin oxidation could have substantial benefit, so we evaluated the ability of a -minosteroid, u a, to inhibit myelin oxidation by monocytes and pmn. fresh rat brain myelin was harvested by multiple sucrose gradient, ultracentrifugation steps, and the final product was confirmed to be pure myelin by sds-page electrophoresis. human monocytes or pmn were obtained from healthy, unmedicated volunteers by gradient separation techniques. monocytes ( . x lo cells/ml) and pmn ( . x lo cells/ml), myelin ( pg protein/ml), and lipopolysaccharide ( pg/ml) were incubated for hours with or without wm u a in -ml wells. myelin oxidation was evaluated by a thiobarbituric acid reactive substance assay for production of malondialdehyde (nmol/ ml). myelin oxidation by monocytes was . +- . (mean standard error of mean) without u a and was reduced to . ? . by pm u a ( p < . ). pmn-mediated myelin oxidation was . _t . without drug and . ? . with drug ( p < . ). these results demonstrate that u a markedly inhibits monocyte-and pmn-mediated myelin oxidation and suggest that the aminosteroids may help disorders associated with inflammatory cell-induced myelin injury. microglia cells participate in the pathological reactions of the cns to multiple insults including trauma, inflammation, and neuronal degeneration. functional roles for these cells could include mediating tissue in jury, promoting repair, or modulating immune responses. with regard to the latter, we have observed that the majority of adult human-derived microglia express major histocompatibility complex (mhc) class molecules under basal culture conditions, in contrast to astrocytes derived from the same surgical biopsy specimens. all morphological subtypes of the microglia (ameboid, bipolar, and ramified) expressed mhc class i molecules, indicating a discordance between morphology and mhc antigen expression as markers of microglia activation. the microglia actively ingest myelin constituents, as assessed using fluorescein-labeled myelin basic protein and laser confocal microscopy. autologous t cells (e') freshly isolated from the systemic blood and cocultured with candida antigen underwent active proliferation in the presence of to % microglia, indicating the functional capacity of the microglia to serve as antigen-presenting cells. y-interferon augmented both mhc class expression and functional antigen-presenting capacity. these results indicate the potential of the adult human microglia to promote immune reactivity within the cns. protein (mbp) neutralize anti-mbp purified from multiple sclerosis cerebrospinal fluid active phases of multiple sclerosis (ms) are associated with increased titers of intrathecally produced antimyelin basic protein (anti-mbp). anti-mbp can be purified by antigenspecific affinity chromatography from csf igg of patients with acute relapses of ms. eighteen synthetic peptides of human myelin basic protein (h-mbp) containing between and amino-acid residues and covering the entire length of the molecule were synthesized by the fmoc method. purified anti-mbp was reacted with increasing amounts of h-mbp as well as each of the peptides in an initial liquid phase assay, and subsequently titers of f anti-mbp in all resulting mixtures were measured by a solid-phase radioimmunoassay . purified anti-mbp was neutralized by h-mbp and of the synthetic peptides containing overall residues corresponding to to of h-mbp. the remaining synthetic peptides covering both the amino and carboxyl terminals of h-mbp did not significantly react with purified anti-mbp from these patients. in conclusion, anti-mbp purified from csf of ms patients has affinity for epitopes located between residues and of h-mbp. in a double-blind study involving patients, we recently demonstrated that -aminopyridine ( -ap) is superior to placebo in the treatment of multiple sclerosis (ms) (ann neurol, in press). the related agent , -diaminopyridine (dap) also appears to be effective. to enable a preliminary comparison, patients, who in our previous study had not benefitted from -ap, were now treated ( wk) with dap (up to . mg/kg/day) for weeks in an open-label fashion. instruments for assessment and registration of side effects were the same as in the previous trial. the optimal dose of dap was . mg/day compared to . mg/day for -ap. significant changes in the edss ( . point or more) were not found, whereas significant improvements in neurophysiological parameters were found (no difference between -ap and dap, allp > . ). subjective side effects during -ap ( patients) mainly suggested cns-function disturbance (dizziness and gait disturbance) and during dap ( patients) mainly suggested peripheral nervous system-function disturbance (paresthesias). systemic tolerability clearly was diminished for dap compared to -ap, with patients withdrawing because of severe gastric complaints and developing liver function abnormalities. these data suggest that -ap is more valuable than dap in the treatment of ms. cy clophosphamide/methylprednisolone therapy in multiple sclerosis multiple sclerosis (ms) is a presumed autoimmune disease in which various forms of immunotherapy have been attempted. mri studies show the disease to be more chronically active than is clinically evident, thus a single treatment is unlikely to provide lasting benefit. recently, the northeast cooperative treatment group found that pulse cyclophosphamide ( mg/m every other month for years) slows progressive ms. we initiated a pilot study to determine the effect of a more intensive and prolonged pulse therapy regimen in both progressive and earlier stages of the disease. pulse therapy was given after induction with either iv cyclophosphamide/corticotropin ( mg/m x over days) or iv methylprednisolone ( gm x over days). patients received a single iv dose of cyclophosphamide ( - , mg/m ) adjusted to produce leukopenia plus gram of iv methylprednisolone monthly for a year, every weeks for the next year, and every months in the third year. another group received pulse methylprednisolone without cyclophosphamide. as of this writing, patients have been treated, of which have completed years. interim analysis shows that patients treated with pulse methylprednisolone were more likely to become treatment failures than those treated with pulse cyclophosphamide/methylprednisolone ( % vs %) independent of induction therapy. withdrawal due to toxicity, however, was higher in the pulse cyclophospharnide group. current regimens involve methylprednisolone induction alone followed by pulse cyclophosphamide/methylprednisolone, analogous to lupus nephritis pulse therapy. this regimen can be given solely on an outpatient basis, does not cause alopecia, and is more amenable for use in earlier stages of the disease. to determine the incidence of pathologically confirmed malignancy in multiple sclerosis (ms) patients in funded clinical trials of cyclophosphamide (ctx) and of azathioprine (aza), data were collected longitudinally using telephone interviews, written questionnaires, physical examinations, and medical records for ctx and aza patients from a community in-hospital and out-patient ms clinic in fargo, nd. in the ctx study (goodkin et al, arch neurol ; : - ) , clinically definite (cd), chronic progressive ms patients were enrolled. twenty-four were controls and received a mean induction dose of . grams. fourteen of the induced patients then received boosters every other month for months resulting in a mean total dose of . grams. no malignancies were detected. in the aza study (goodkin et al, neurology ; : - ) , c d relapsing ms patients participated. twenty-five were controls and received mgtkg of aza daily by mouth adjusted to maintain a white blood count of greater than , o/cmm for years (mean dose . mg/kg). two of aza patients developed resectable skin cancers: basal cell (bcc) at months and i squarnous cell (scc) at months. the incidence of developing a bcc or scc was not significantly increased after initiating therapies as compared to the untreated ms controls (aza: fisher exact testp value = . ). no other malignancy has been detected during to months of clinical follow-up. allergic encephalomyelitis paula dore-dufly, ruth washington, and robert h. swanborg, detroit, m i postcapillary endothelium at sites of inflammation undergoes many changes referred to as activation. activated endothelial cells (ec) exhibit increased surface expression of immunorelevant proteins (icam- ; ncam, elam, and mhc class i and class i antigens [ags)). the sequence of events that characterizes ec activation may be important in susceptibility, induction, and perpetuation of experimental allergic encephalomyelitis (eae). in this study we examine expression of ec activation antigens in central nervous system (cns) microvessels in response to interferon gamma (ifn-y). cns microvessels from sjl and bio.s mice were incubated for hours in ifn-y ( u/ml), fixed, permeabilized, and then stained with an antibody that recognizes class i, class i mhc antigens, icam- , and factor viii. relative fluorescence intensity was determined using a laser cytometer. results indicate that microvessels from all strains tested expressed no detectable icam- and class i ags. little class i antigen and transferrin receptors were expressed. upon stimulation with ifn-y, sjl microvessels exhibited increased surface expression of all ec activation ags. b o.s microvessels exhibited icam- and class i mhc but mhc class i ags were not upregulated. results indicate that there are strain differences in the ec response to ifn-y. resistance of b o.s mouse ec to activation by ifn may be a factor in decreased susceptibility or induction of eae, or both. protein-t-cell line-mediated experimental to investigate a possible pathogenic role of interferongamma (ifn-y) in experimental allergic encephalomyelitis (eae), an immunocytochemical study was undertaken to localize this cytokine in the spinal cord of lewis rats in which eae was produced by adoptive transfer of myelin basic protein-specific t cells. one pm-thick cryosections of spinal cord were labeled with monoclonal antibodies (mab) db- and db- recognizing different epitopes of rat ifn-y. in the spinal cord of naive rats, mab db-i, but not db- , stained processes of astrocytes, suggesting that astrocytes contain a protein with an epitope cross-reacting with ifn-y. in rats with at-eae, numerous ifn-y-positive cells stained with both mab db- -and db- -positive cells were present from days to after cell transfer and had disappeared on day . at day they entered the spinal cords predominantly through subpial vessels. ifn-y-positive cells could be identified as w / + leukocytes as well as ed -positive macrophages. as in naive rats, astrocytes in at-eae were labeled only with mab db- , but not db- . we never observed labeling of motor neurons with these mab. the transient presence of ifn-y in the rat spinal cord at the onset of at-eae suggests a pathogenic role of this cytokine in acute immune-mediated demyelination of the cns probably as a local stimulus for expression of mhc class i antigens and adhesion molecules, as well as for the release of tnf-a and toxic oxygen radicals from macrophages and microglia. immune responses to stress or heat shock proteins are implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis (ms). we examined the hypothesis that antigens in myelin cross-reacted with stress protein antigens. two techniques were used: immunocytochemistry and western blotting. frozen histological sections were prepared from normal human central and peripheral nervous system tissues. sections were incubated with murine monoclonal antibodies to different mycobacterial stress proteins. antibody binding was determined using avidin-biotin complexed antimurine antibody linked to alkaline phosphatase. program and abstracts, american neurological association a monoclonal antibody to the stress protein sp from m . leprae strongly stained both central and peripheral nervous system myelin. no myelin staining was noted with antibodies to sp or sp . proteins from purified central and peripheral nervous system myelin were separated by sds-page. western blots were prepared using a monoclonal antibody to sp and a polyvalent rabbit antibody to myelin basic protein (mbp) as primary antibodies. antibody binding was determined using antimurine or antirabbit igg antibody coupled to alkaline phosphatase. strong staining of central but not peripheral mbp by the anti-sp antibody was observed. the rabbit anti-mbp antibody stained both central and peripheral nervous system mbp. the presence of antigenic epitopes shared by a stress protein and the potential autoantigen, mbp, supports the hypothesis that immune responses to stress proteins may be involved in the pathogenesis of presumed autoimmune diseases such as ms. ( ). of patients with borderline or positive csf lyme titer, received parenteral ceftriaxone. all had later relapses consistent with ms. one patient received a month of oral doxycycline. the fifth patient had a negative western blot and was not treated. we conclude that an incidental borderline or positive lyme serology in an ms patient is unlikely to indicate neurological lyme disease. borderline serologies should be documented to rise on later testing; positive serologies should be confirmed by retest in a different laboratory or by western blot. csf abnormalities suggestive of neurological lyme disease (pleocytosis, protein elevation, intrathecal lyme antibodies) are distinct from those suggestive of ms (ogb, elevated igg index, mbp). in such patients antibiotic treatment may be appropriate, but will not alter disease course. when designing and analyzing therapeutic trials for multiple sclerosis (ms), investigators commonly compare the proportion of patients in the experimental and control groups who worsen one or more steps on the disability status scale (dss) or expanded dss during the study (typically years' duration). however, the intervals between the scores in the dss may not be equal. it may be easier to change by one or more steps in the lower end of the scale (e.g., dss = - ) than in the midportion of the scale (e.g., dss = - ). to evaluate this possibility, we compared the proportion of patients who worsened by one or more steps in the years after entering our program with dss = or with dss = . fifty-one percent ( ) natural history data may be useful for designing therapeutic trials for multiple sclerosis (ms). since , we have collected such data in a standard format on patients in the ucla multiple sclerosis research and treatment program. t o describe the course in our group, we have performed survival analysis (kaplan-meier) of patients with or more assessments who entered the clinic with disability status scale (dss) scores of to (n = ). an increase of one or more steps in the dss score persisting for more than months defines worsening. median times to worsening for a dds at entry of to were approximately years (range . - . ); but were . years for dss and . years for dss . for those starting at dss (n = ), only % worsened by year, % by years, and % by years. the percent worsening when starting at dss (n = ) were %, %, and %, respectively. these variable rates of worsening (i.e., time spent at each starting level) influence therapeutic trial design. including patients with dss or will increase the sample size and study duration. for testing nontoxic agents, we recommend enrolling patients with dss to . for more toxic treatments, we suggest dss to . (partially supported by usphs grant ns , the conrad n. hilton foundation, and various donors.) pi . cholinergic antagonists and p-adrenergic agonists inhibit experimental allergic encephalomyelitis in an additive manner mark a. jensen, avertano noronha, and bawy g. w. amason, chicago, il lymphoid organs receive a sympathetic (sns) and possibly a parasympathetic innervation. lymphocytes express padrenergic and cholinergic receptors and thus are sensitive to regulation by these neurotransmitters. the severity of experimental allergic encephalomyelitis (eae) is increased in sns-ablated animals. local parasympathectomy decreases plaque-forming responses in submandibular nodes. p,-adrenergic receptors are upregulated on cds t cells in progressive multiple sclerosis, as are m,-muscarinic acetylcholine receptors on cd t cells. we examined the effect of isoproterenol, a p-adrenergic agonist, and scopolamine, a cholinergic antagonist, on the course of eae in lewis rats. eae was induced by injection of . ml of incomplete freund's ad juvant containing guinea pig spinal cord ( % wlv) and m. tubercdosis ( mgiml) in one hind footpad. scopolamine ( . mglkg, twice daily) and/or isoproterenol(o. mglkg, twice daily) or saline were injected subcutaneously starting on the day of immunization. scopolamine or isoproterenol alone reduced severity ( p < . , t test) and duration ( p < . , t test) of disease compared to controls. the combination of scopolamine and isoproterenol further reduced disease severity compared to either agent alone ( p < . , x test), suggesting an additive protective effect of cholinergic antagonists and p-adrenergic agonists in eae. antigen-presenting cells a . conrad, v. sanders, p. schmid, and w. w . tourtellotte, los angeles, c a tissue is cryopreserved by the method of tourtellotte; this procedure minimizes or eliminates ice artifacts and preserves surface protein markers. the dissected plaques are lightly fixed in % paraformaldehyde and then suspended in % sucrose. blocks are mounted in oct, cryosectioned at p,m, and picked up on gelatinized slides. the activity of plaques is determined by the presence or absence of myelin debris (antimyelin basic protein stain or lux fast blue) or presence or absence of neutral lipids indicating myelin digestion as seen by oil red (oro) staining and the presence or absence of a class i major histocompatibility complex antigen (evidence for an antigen-presenting cell) on macrophages or microglia as seen by immunocytochemically staining for hla-dr (hb atcc). the following is our classification of the activity of multiple sclerosis (ms) plaques. type i, the most active, is defined as an area of hypercellularity, positive for hla-dr with no o r staining or staining for myelin debris. type , or active, is defined as an area of hla-dr-positive cells that stain mildly with o r at the plaque edge but positive for myelin debris; evidence for demyelinating activity < hours (prineas; raine). there is an inner area of plump cells that are positive for hla-dr and oro. type , or modestly active, is defined as a "shelf" of plump hla-dr-positive cells at the edge loaded with program and abstracts, american neurological association oro-stained neutral lipid but a paucity of or no myelin debris. a region of hypocellularity is observed at the center of plaque. type iv, least active or inactive, is defined as scattered hla-dr-positive cells at plaque edge with little or no o r staining and no evidence of myelin debris. the frequency of plaque types was determined from a random sample of ms tissue blocks from patients who died of ms. ten percent of the plaques were type i; %: were type ; % were type . the majority of plaques ( %) were inactive (type iv). accordingly, it is necessary to classify the demyelinating activity of ms plaques for protocols designed to investigate etiopathogenesis. do these results suggest that whatever causes ms can be eradicated in half of the demyelinating areas by the time of death? p . localization of g d l b ganglioside antigen in the human peripheral nervous system susumu kusunoki, atsuro chiba, tadashi tai, and lchiro kanazawa, tokyo, japan serum antibodies against ganglioside gm and/or g d l b frequently are detected in autoimmune neuropathies such as rnultifocal motor neuropathy, igm paraproteinemic neuropathy, and guillain-barre syndrome. some of them bind to gm or g d l b monospecifically but the others cross-react with both of the antigens. to investigate respective localizations of gm and g d l b antigens in the human peripheral nervous system (pns), immunohistochemical study of dorsal root ganglia (drg), dorsal roots, ventral roots, and sympathetic ganglia (sg), which were obtained from human autopsy specimens, was performed by using mouse monoclonal antibodies, each monospecific to gm and gdlb. ggr , monospecific to gd b, immunostained nerve cell somas and axons of drg, sg, and dorsal and ventral roots. ggrl also recognized some myelin, including paranodal areas. however, gmb , monospecific to gm , did not bind to either neuron or myelin. thus, serum anti-gdlb antibody can bind to neurons and some myelin in the human pns. further study is necessary to identify the localization of gm antigen, because previous biochemical studies have shown that gm is also present in the human pns. the purpose of this study was to compare t and t lymphocytes in migraine patients versus controls, and a review of the literature was done. fifty-six migraine patients, aged to , were tested, as were controls. the t :t ratio in migraine patients was : , compared to : for controls. suppressors (t ) were reduced from in controls to in migraine patients. helpers (t ) decreased from , in controls to in migraine patients, and total ?' lymphocytes decreased from , to , . previous studies have revealed similar differences in t lymphocytes, with higher t : t ratios being found in both migraine and tension-type headache patients. in food-induced migraine, an increase in circulating immune complexes was noted, with increased t levels. differences in serotonin binding to mononuclear cells have been noted in migraine patients. a decreased sensitivity of the lymphocyte beta-adrenergic receptor in migraine patients was suggested by one study. after lymphocyte incubation with il- , the natural cytotoxic response is augmented in cluster patients. the percentage of lymphocytes expressing receptors for il- was decreased in cluster patients in a fur-review of the literature ther study. this il- receptor defect was independent of whether the clusters were present. there is a loss of highaffinity binding sites for serotonin on lymphocytes in both episodic tension and chronic tension headaches. we used positron emission tomography (pet) to measure local cerebral blood flow in volunteer subjects while they performed tasks of memory-guided saccades and a visual fixation control. tasks were performed continuously for seconds during emission scans, after bolus injection of h lso. eye movements were verified with electrooculography. areas of significant increase in regional blood flow between tasks were matched to three-dimensional reconstructions of brain magnetic resonance images of each subject. compared to the visual fixation control, saccade tasks evoked bilateral activation in the posterior superior parietal lobule with extension into the inferior parietal lobule. activation was also seen bilaterally in an area of frontal cortex immediately rostra to the precentral gyrus extending from the superior frontal gyrus to the inferior frontal sulcus. the increased blood flow in the posterior parietal cortex most likely corresponded to enhancement of visual attention required during saccades, while that in the frontal lobe, which included the frontal eye fields, indicated activation for saccade motor output. pamela blake, alexander s. mark, martin kolsky, and jorge kattah, washington, dc fifty patients with third cranial nerve (cn) palsy underwent precontrast and postcontrast mri to assess the utility of this study in this clinical context. mri demonstrated an appropriate lesion in cases. six patients had brainstem lesions ( infarcts, mass lesion, cryptic vascular malformation, hemorrhagic shearing injury, with compound q toxicity). lesions of the cisternal segment of the nerve were present in patients ( aneurysms, lymphomas, ophthalmoplegic migraine, viral meningitis, coccidiodomycosis, nerve avulsion), with enhancement of this segment in patients. fourteen patients had cavernous sinus lesions ( lymphomas, nasopharyngeal carcinoma, tolosa-hunt syndrome, cavernous carotid aneurysms, pituitary apoplexy, aspergillosis). eighteen patients, all with history of diabetes or vascular disease, had normal mri results, suggesting microvascular infarction of c n . in patients with cn i palsy, mri can detect the presence of brainstem or cavernous sinus lesions and often can suggest their cause. mri with contrast enhancement can demonstrate involvement of the cisternal segment of cn i in patients with inflammatory or infiltrative processes that previously could not be radiographically demonstrated. our study suggests microvascular infarction does not cause nerve enhancement on contrast-enhanced mri. we describe patients with acute hyperglycemic, hyperosmolal, nonketotic stupor who had ocular flutter or opsoclonus clinically. these are the fourth and fifth adult patients reported with the acute onset of stupor and opsoclonus; all patients had nonketotic hyperglycemia and hyperosmolality (rapid eye movement sleep also causes stupor and saccadic eye movements). three of the patients had myoclonic jerks in addition to opsoclonus. in the , opsoclonus began when glucose and osmolality acutely increased, and completely resolved when glucose and osmolality became normal, showing that opsoclonus is a specific and reversible effect of the metabolic disorder and implying that either acute hyperglycemia or hyperosmolality directly causes opsoclonus. since acute hyperosmolality caused by nacl or sucrose can cause a similar syndrome in experimental animals (trans am neurol assoc ; : - ) and infants, hyperosmolality is probably more important. opsoclonus is thought to be due to abnormal activity of saccadic "burst" cells in the pons. acute hyperosmolality may cause spontaneous saccades by disinhibiting burst cells from normal "pause" cell inhibition or by directly activating burst cells. the combination of acute deterioration in mental status and either ocular flutter or opsoclonus should suggest acute hyperosmolality, in particular, nonketotic hyperglycemia. neural cell adhesion molecule (n-cam) and the related cell adhesion molecule l play significant roles in axon outgrowth and mediation of cell-cell contact in development, and after peripheral nervous system injury. to understand the role of these molecules after injury in the adult cns, we studied alterations in n-cam and l in the rat brain's response to entorhinal cortex (erc) lesion. post lesion, reactive synaptogenesis and axonal sprouting follow a well-defined temporal course in restoring the synaptic density of the dederented outer two-thirds of the hippocampal dentate gyrus molecular layer (ml) to near prelesion levels. we found striking regionand lamina-specific staining of n-cam and l in the normal hippocampus and marked alterations in these molecules after injury. embryonic n-cam, present in high amounts during development but expressed at very low levels in the adult hippocampus, was massively re-expressed in the denervated zone; the embryonic form was still heavily expressed days later, when synapse number returned to >so% of prelesion levels. l staining, normally evenly distributed through the ml of the dentate, was completely lost in the outer ml, which is denervated by the erc lesion. this staining had not returned by days after lesion. neural cell adhesion molecules play a role in specificity of neural connectivity both in development and after injury. in reactive synaptogenesis and axonal sprouting after injury, both ontogenetic (the reexpression of embryonic epitopes) as well as uniquely adult sequences of repair are utilized. following hemispherectom y" a. pascual-leone, h . t. chugani, l. g. cohen, j . p . brasil-neto, e. m . wassermann, j . and m. hallett, betbesh, md, and los angeles, ca we studied subjects, aged months to years, who underwent hemispherectomy months to years earlier for intractable epilepsy. all had a spastic hemiparesis contralateral to the resected hemisphere, which was present presurgically. we used focal transcranial magnetic stimulation to map the areas of the preserved hemisphere targeting the abductor pollicis brevis (apb), the biceps, and the deltoid ipsilaterally and contralaterally. in subjects who had hemispherectomy after age , the same area targeted ipsilateral and contralateral muscles. in the remaining subjects, who were more functional, areas targeted ipsilateral muscles. one area coincided with the contralateral representation, but stimulation induced motor-evoked potentials (meps) of lower amplitude and longer latency in the ipsilateral muscles. the other area, to cm anterolaterally, targeted exclusively ipsilateral muscles and stimulation induced meps of normal amplitude and latency. this separate ipsilateral representation was more distinct in subjects studied a long time after the hemispherectomy and in those who were younger at the time of the operation. these results show evidence of motor reorganization after hemispherectomy. better motor function is associated with topographically differentiated ipsilat-era and contralateral representations, which may depend on age at the time of hemispherectomy and the time since then. cynthia l. comella, glenn t . stebbins, nancy brown-toms, and christopher g. goetz, chicago, il in a single-blind, crossover study, we evaluated the effect of an intensive outpatient physical rehabilitation program (re-hab) on the severity of parkinson's disease (pd). the re-hab program consisted of -hour sessions per week for weeks. sixteen patients completed phases, a rehab phase and a control phase, separated by months. the order of participation in each phase was randomized. all patients were evaluated using the unified pd rating scale with subscales for mentation (ment), activities of daily living (adl), and motor function (mot) by an investigator blinded to the rehab phase of the patient. all patients were evaluated immediately before and after each phase. pd medications were not changed during any phase. following the re-hab phase, there was significant improvement in adl score (pre-rehab , post-rehab , p = . wilcoxon) and in mot score (pre-rehab , post-rehab , p = , wilcoxon), but no change in ment score. after the con-trol phase, there was no significant change in any outcome measure. this is the first controlled, crossover study of an intensive rehab program in pd. it demonstrates that re-hab improves objective motor function and adl scores. sensitive and quantitative measurements of leg weakness, one of the most common deficits in multiple sclerosis (ms) patients, can be made using specialized extremity testing equipment. to determine whether such determinations could be used in clinical trials, ms patients with leg weakness that had been stable for at least months were evaluated every days over a -month period. each testing session was carried out at the same time of day and medication dosages and schedules were kept constant (only patient was taking baclofen and his dosing remained constant). quadriceps and hamstrings strengths were measured in isometric contraction in a mechanical testing apparatus (kincom). variability for each series of determinations was expressed as the standard deviation of the mean as a percent of the mean. the overall variability then was expressed as the mean of the individual variabilities. the mean variability for the strength determinations over months was . k . , and only one series of determinations out of had greater than % variability. these results suggest that quantitative strength determinations might be useful in clinical trials, and early experience in trials of aminopyridines will be discussed. polymyositis (pm) is an inflammatory myopathy of unknown cause, but the accumulating data strongly suggest an autoim-mune pathogenesis. the histological picture is of muscle fiber necrosis and inflammation, whereas in postviral fatigue syndrome (pfs), a disorder characterized by severe fatigue with myalgia and psychiatric symptoms, the histological picture of muscle is essentially normal. enteroviruses have been implicated on epidemiological and serological studies in both. we have used the polymerase chain reaction (pcr) and an enteroviral-specific probe and found persistent enteroviral genomic material in both pm and pfs muscle biopsy specimens. furthermore, we used a radiolabeled full-length cdna probe derived from coxsackie b in an in situ technique to look for viral d n a in pcr-positive cases. coxsackie genome was clearly identifiable in the muscle biopsy specimens of patients with pm but negative in pcr enteroviral-positive cases of pfs. the virus excites an inflammatory reaction only in pm. a murine animal model for pfs developed in our laboratory showed positive muscle pcr using enterovirus probes and a conspicuous increase in interleukin within the brain. these results provide major clues in the search for the etiology of these two puzzling disorders. human t-lymphotropic virus type i (htlv-i) is a cause of adult t-cell leukemia and tropical spastic paraparesis. in a specific population of iranian jews originating from the city of mashad, there is a high incidence of htlv-i infection ( . %) and associated t-cell leukemia. we evaluated the incidence of possible correlation between htlv-i infection and spastic paraparesis in israeli mashadi-born jews. we have examined mashadi-born immigrants in a mashadi community center ( men, women, mean age t_ . yr) and non-mashadi iranian-born jews. blood samples were tested for htlv-i antibodies by particle agglutination test. the polymerase chain reaction (pcr) was used to amplify htlv-i sequences of d n a from peripheral blood mononuclear cells. twelve mashadi-born immigrants ( %) were seropositive for htlv-i. in of those serologically positive for htlv-i ( %), neurological examination revealed spastic paraparesis of varying severity. none of the non-mashadi iranian jews were seropositive for htlv-i or had clinical signs of spastic paraparesis. these results support other studies of htlv-i-associated myelopathy. the high incidence of htlv-i-associated spastic paraparesis in the mashadi community might be related to their unique history of a high rate of intermarriage among members of this ethnically segregated group. further epidemiological studies are underway to evaluate the incidence of htlv-i in mashadi families as well as in mashadi-originating jews born in israel, to identify whether infection might be genetically transmitted. lymphotropic virus t y p e i-associated acute t-cell leukemia/lymphoma william j . harrington, jr, william a. sheremata, susan snodgrass, and mark raven, miami, fl human t-cell lymphotropic virus type i (htlv-i)-associated acute t-cell leukernia/lymphoma (atl) is thought to produce important c n s disease infrequently. we wish to correct this impression by presenting neurological findings in patients seen between and the present. all had positive western blots to htlv with polymerase chain reaction confirmation of htlv-i infection. only had a concomitant human immunodeficiency virus infection. all were black, aged to years, were men and women. all but americans came from the caribbean nations of haiti ( , dominican republic (l), jamaica ( ), and trinidad ( ). sexual transmission was the risk factor for htlv-i for all except for intravenous drug user. all patients were systemically ill. three had preceding neurological abnormality ( , months, and yr) and had major neurological disease concomitantly. two of these had tumor masses demonstrated in brain and in the spinal canal. one had a minor facial sensory abnormality; only had no deficits. we conclude that cns disease is commonly associated with atl, and that in the us atl occurs principally in caribbean natives. a. nath, v . hartloper, and m . furer, winni$eg, manitoba, canada microglia and astrocyte cultures were established from human fetal brain. microglia were infected with human immunodeficiency virus (hiv) strains, hiv,, or hiv,,,, resulting in a rising titer of p antigen in the supernatants. multinucleated giant-cell formation, vacuolar changes, and rising levels of lactate dehydrogenase in the supernatants were seen, indicating a cytopathic infection of microglia. astrocytes were infected with free virus or cocultivated with an hiv-infected lymphocyte cell line (hut- ). after a productive phase (rising titers of p antigen and detection of hiv antigens by immunocytochemistry), the cells went into a latent phase where hiv could be detected only by dna polymerase chain reaction. a -fold increase in astrocytes staining for hiv antigens was seen after cocultivation with hut- cells. lymphocytes adhered to astrocytes by hours of cocultivation. no adhesion was seen to microglia. fusion of plasma membranes was seen on electron microscopy. infected astrocytes did not show cytopathic or morphological changes. cell-to-cell contact may be important in viral transmission to astrocytes. hszao-huei chen, steven b. stein, wing kong, and raymond p. roos, chicago, i l one of our goals is tq delineate molecular determinants for disease phenotypes produced by theiler's virus (tv), a mouse picornavirus. the identification of these genes and gene products may clarify viral pathogenesis and also lead to the identification of genes that are important in normal cns function and nonviral cns disease. members of the gdvii subgroup of tv cause an acute, fatal neuronal infection, whereas members of the to subgroup are less neurovirulent and produce a demyelinating persistent infection. our studies of infectious tv cdna clones have demonstrated that the gdvii b(vp )- c segment is critical for neurovirulence. several other areas of the genome, including the ' untranslated region (s'utr), also affect tmev-induced disease. the 'utr of poliovirus, another picornavirus, has a critical role in paralysis; this effect on neurovirulence is believed to result from an altered translational efficiency related to bind-region ing of neural cell proteins. tv 'utr has an unusual predicted secondary structure, even for picornaviruses. our studies demonstrate that the tv 'utr affects translational efficiency and has a distinctive protein-binding pattern. investigations of the tv 'utr may clarify features of translational regulation of cns genes in general. p . coronaviruses infect primate brain from g a y f. cabirac, ronald s. murray, galen cai, kristen hoel, and kenneth soike, englewood, co, and covington, la recently, we described finding coronavirus (cv) rna and antigen in active demyelinating plaques of multiple sclerosis (ms) brain tissue (murray et al, ann neurol, in press). molecular analysis showed the cv rna to be more closely related to murine cvs than to human cvs. we then demonstrated that, following intracerebral inoculation, the murine cv jhm and the putative ms isolate cv-sd could infect and cause demyelination in primate brain (murray et al, virology, in press). we now have data showing that murine cv can infect primate brain following intranasal or intravenous routes of inoculation. standard virology, histopathology, and the molecular analysis of viral cytotropism will be presented. we conclude that cvs related to murine cvs can infect primate cns from peripheral routes and warrant consideration as potential human pathogens. probes (gag, pol, or enw). eleven were white and were black. a history of transfusion was obtained in , and sexual risk of transmission was present in but not in others. fulminant disease occurred in transfused men months to years later but such disease was only seen in woman (in month). in contrast, of women with multiple sexual partners had rapid progressive disease. the male-to-female ratio was : for transfusion association and : for those at sexual risk but : for unknown risk, transfusion is an important risk for tsp/ham and the diagnosis must be considered in all gait problems, regardless of the "diagnosis."transfusion association should decrease with regular testing of blood donors, but this will not affect the risk of sexual transmission. spastic paraparedhtlv-i-associated myelopathy (tsp/ ham). it is unclear why htlv-i infection causes atl in some individuals and tsplham in others. differences in the genome of viral isolates or immunological and host factors have been hypothesized to play a role in disease expression. at present there are no animal models of tsplham and no suitable animal models of htlv-i infection that can address these issues. we transplanted severe combined immunodeficient (scid) mice with peripheral blood mononuclear cells (pbm) from tsplham patients in an attempt to produce htlv-i disease. two weeks after transplantation of pbm from tsplham patients, we detected anti-htlv-i igg in serum samples of of scid mice by enzyme immunoassay using sonicated whole virus. five weeks after transplantation, we detected anti-htlv-i igg to p , p , gp , or gp in serum samples of of scid mice by immunoblot of disrupted virus. these findings suggest that scid mice may be valuable in the study of molecular and pathological determinants of htlv-i-induced disease. theiler's virus produces an encephalomyelitis in susceptible mice. during the course of the disease, specific regions in the cns become infected. compared to the immunocompetent mouse, the nude mouse provides a useful model where viral dissemination can be studied in the absence of functional t lymphocytes and antibodies. we investigated the distribution and spread of the da strain of theiler's virus in the cns of nude mice. by immunohistochemistry, the hippocampus, arnygdaloid nuclei, entorhinal cortex, cingulate cortex, thalamus (anteroventral nuclei), midbrain, and spinal cord all contained viral antigens by weeks after infection. in addition, the olfactory nuclei, mamillary body, hypothalamus, basal ganglia, and nucleus raphe dorsalis often were involved. in the brain, the limbic system was the site commonly infected by theiler's virus. the time course of virus dissemination varied depending on the site of initial virus infection, though the final distribution of virus was the same. olfactory bulb injection, which is a direct inoculation into the olfactory pathway, resulted in more rapid spread than did cortex injection. we demonstrated the constant presence of viral antigen in the limbic system and a different kinetics of viral dissemination between the two different routes of intracerebral inoculations. these results suggest that limbic structures and their connections are important to the dissemination of theiler's virus. -associated myelopathy in a northwest native indian d. foti, d. werke, g. dekaban, g. p. a . rice, andj. oger, vancouver, bc, and london, ontario, canada a -year-old indian of the oweekeno tribe was admitted for investigation of a myelopathy. initially symptomatic with midthoracic radicular pain, she developed progressive spastic paraparesis over year with mild sensory symptoms and urinary retention. mri of the cord and brain were normal. csf showed elevated protein ( mgll), lymphocytes, and weak oligoclonal banding with evidence of intrathecal igg synthesis. antibodies to human t-lymphotropic virus type i (htlv-i) were positive by enzyme-linked immunosorbent assay and western blot on both serum and csf. presence of htlv-i was demonstrated by polymerase chain reaction of blood lymphocytes. htlv-i-associated myelopathy, or tropical spastic paraparesis, is endemic in southern japan, the caribbean basin, and several tropical islands but has not been reported in natives of northwest canada. this patient's only risk factors for htlv-i infection were blood transfusions years previously. ongoing familial and epidemiological studies as well as virus sequencing should indicate if this case represents an indigenous or an imported infection. caused by herpes simplex virus type lawy blankensbz) and herbert . newton, columbus, oh myeloradiculitis occasionally occurs secondary to herpes simplex virus type (hsv ) infection, but rarely has been reported after herpes simplex virus type (hsv ) infection without encephalitis. we describe a -year-old man who developed cervical myelopathy and radiculitis, never developed symptoms of encephalitis, and had positive hsvl spinal fluid cultures. h e initially developed extremity weakness and incoordination, numbness, paresthesias, and neck pain. evaluation was negative except for mri results, which showed a high-signal lesion centrally within the cervical cord. the weakness, sensory loss, and radicular pain progressed over several months. subsequent mri showed extension of the high-signal abnormality and mild enlargement of the cervical cord. symptoms stabilized briefly with dexamethasone but soon worsened, and were accompanied by paroxysmal kinesiogenic dystonic episodes of his arms and right leg. repeat evaluation was unrevealing except for the spinal fluid, which grew out hsv . the patient was treated with dilantin and a i-month course of intravenous acyclovir, with slow improvement of neurological status and resolution of the dystonic episodes. this case illustrates that hsvl can cause a myelopathy with a subacute and protracted course, requiring serial was more frequent in the middle-aged group ( p < . , p = . ). history of hypertension, previous strokes, diabetes mellitus, and antithrombotic treatment was similar, as were sex ratio, qualifying event type (transient ischemic attack or nondisabling stroke), and angiographic features; stenosis severity in either side and presence of ulceration were all similar. in elderly patients, ecad is associated with symptomatic cardiac disease (scad or af), whereas in middleaged patients it is associated with precursors of generalized atherosclerosis (smoking and hyperlipidemia). to identify the anatomic factors correlating with dementia in patients with lacunar infarctions, we examined digitized ct data on elderly patients (mean age = . yr; education = yr) who presented with acute lacunar infarction. dementia was diagnosed in patients ( . %) based on neuropsychological tests given months after stroke onset. the following ct variables were assessed: infarct location and number, total infarct volume, brain parenchymal and csf areas at levels, and width of the frontal horn, third ventricle (tvw), and lateral ventricle plus their ratio to the intracranial width. atrophy and leukoaraiosis were rated semiquantitatively using a standard scoring method. in the group overall, mean infarct volume was . cc and mean infarct number was . . in univariate analyses, dementia was significantly related to infarct volume, number, tvw, bilaterality, and infarct predominance on the left side, but not to leukoaraiosis or atrophy. in a regression model adjusting for demographic factors, the ct variables correlating independently with dementia status were infarct number (p = . , p = . ) and the presence of left cerebral infarcts (p = . , p = . ). we conclude that the most important ct variables related to dementia in lacunar stroke are lesion multiplicity and the presence of lesions on the left side. brain ischemia results in potassium (k+)-induced voltageregulated presynaptic calcium (ca") accumulation, which may contribute directly to neuronal in jury presynaptically, and also promote excessive release of excitatory neurotransmitters leading to cell damage postsynaptically. k+-induced depolarization of brain synaptosomes may be used as an in vitro model to study the therapeutic potential of pharmacological agents to alter ischemia-induced presynaptic ca + accumulation. we preincubated gerbil cerebral cortical synaptosomes in r (janssen pharmaceutical) at concentrations of to lo-' m, subsequently depolarized the synaptosomes with k + , at concentrations of to mm, and measured intrasynaptosomal ca ' ([ca +]) with the fluorescent indicator fura . r had no effect on ecaz'i in nondepolarized synaptosomes, but significantly (<. , student t ) depressed depolarization-induced {ca +] at to lo-' m in a dose-dependent fashion. the greater the degree of depolarization employed, the greater degree of depression in [ca +] was seen at each dose of r . since r had no effect on [ca +] when utilizing ca +-free incubation media, it was demonstrated that r prevents depolaritation-induced [ca ' ] increase by blocking voltage-regulated influx. because r appears to block voltage-regulated presynaptic ca ' accumulation, it should be further evaluated as a potential therapeutic agent after cerebral ischemia. sneddon's syndrome (ss) is a focal and diffuse arthropathy affecting mainly the vascular wall of the skin and cerebral arteries. etiology is not determined. we studied patients with ss ( females, males), aged to years. clinical, radiological, and immunological studies were done. all patients developed cerebrovascular disorders: ischemic stroke in %, transient ischemic attack (tia) in %, and ischemic stroke and tia in %. cerebral scan showed small and medium (less than cm) ischemic lesions in %. these lesions were superficial in the cerebral cortex ( %); deeper in the centrum semiovalis, internal capsule, and basal ganglia ( %); and both superficial and deeper ( %). ultrasound and angiogram studies revealed obstruction of intracranial arteries in %, and of extracranial arteries in %. partial or complete improvement of cerebrovascular symptoms was observed in %. immunological studies showed increased content of b-cell lymphocytes ( p < . ), increased levels of igm ( p < o.oool), and circulating immunocomplexes ( p < . ). anticardiolipin antibodies were increased ( %) and lupus anticoagulant detected ( %). cerebrovascular disorders in ss that lead to small ischemic cortical lesions have a good prognosis. pathogenesis of this syndrome may be related to antiphospholipid antibodies. the cheiro-oral syndrome is characterized by pure sensory deficit limited to the hand and mouth. this syndrome has been described in diverse lesions of the parietal operculum and brainstem, but occurs most commonly due to lesions of the contralateral thalamus, and suggests a humuncular representation of sensation in the ventral posterior lateral (vpl) and ventral posterior medial (vpm) nuclei. we describe a -year-old hypertensive woman who presented with sudden onset of numbness of the left side of her body. examination revealed a left hemisensory deficit to all modalities that spared the hand and peri-oral regions. cat scan and mri demonstrated an acute infarction of the posterolateral right thalamus, with a rim of preservation adjacent to the internal capsule. pure hemisensory loss with sparing of the hand and mouth ("inverse cheiro-oral syndrome") has not been reported previously, and complements previously published studies of the cheiro-oral syndrome in demonstrating somatotopic sensory representation in the thalamus. to examine the relationship between depression and dementia after stroke, we administered the -item hamilton depression rating scale (hdrs) and neuropsychological tests to elderly patients months after ischemic stroke. using dsm- -r criteria, we found dementia in ( . %). hdrs score was . -t . overall, and higher in demented compared to nondemented patients ( . . vs . t . , p = . ). the frequency of depression (total hdrs score > ) was also higher with dementia ( . % vs . %, p = . ). however, demented patients did not differ from nondemented patients on ratings of depressed mood. instead, hdrs items for psychomotor retardation, reduced work activities, and impaired insight best distinguished the groups. in multiple regression analysis, stroke severity (p = . , p = . ) was the most important correlate of hdrs score; dementia status was not correlated independently. mean scores on mini-mental state examination and neuropsychological tests assessing memory, orientation, verbal, spatial, attentional, and abstract reasoning skills did not differ by depression status. although weakly related to intellectual impairment, hdrs score in our stroke sample was most importantly associated with stroke severity. higher scores on hdrs in demented stroke patients may be explained by physical and cognitive symptoms that are expected with dementia. these findings do not support a causal link between depression and dementia, and argue against the importance of "depressive pseudodementia" as an explanation for intellectual decline after stroke. to investigate the pathophysiological mechanism of the white matter lesions in progressive subcortical vascular encephalopathy (psve) of binswanger type, we measured regional water partition coefficient (pc) (reflecting water content) and effective p h (pht) (weighted average of intra-and extracellular ph) with dynamic positron emission tomographic technique using - co,, o,, and c- co,. si-multaneously, regional cerebral blood flow (rcbf) and regional cerebral metabolic rate of oxygen (rcmro,) were evaluated. eight subjects ( normal, psve, multiple infarction) were examined. in the white matter lesions in psve, corresponding to high-intensity areas in t -weighted images of mr, the pc increased and pht was unchanged or decreased, whereas both rcbf and rcmroz declined. the ratio of white matter pc to gray matter pc was . in psve and . in normals. in the frontal gray matter, the pc decreased in psve, whereas rcbf and rcmroz also decreased. these results suggest that tissue water content increases in the white matter lesions in psve reflecting the edematous status of the damaged regions. elevated pht with high pc may reflect the increase of extracellular water of the tissue. measurement of pc and pht provides useful information about the pathogenesis of psve. stroke has been the second most common cause of death in korea but its risk factors (rfs) have not been studied intensively, so the rfs or causes were investigated prospectively in , consecutive stroke patients who were admitted to the chungnam national university hospital, taejon, korea, between and . they included cases of cerebral ischemia (ci) ( . %), cases of intracerebral hemorrhage (ich) ( . %), and cases of subarachnoid hemorrhage (sah) ( . %). control data were obtained from healthy spouses of the patients. multivariate analyses showed that hypertension (ht) was the strongest rf for all stroke types and was followed by old age, diabetes mellitus (dm), smoking, high-density lipoprotein cholesterol, and fibrinogen. the rfs for atherothrombotic ci included old age, ht, dm, smoking, fibrinogen, and cholesterol. for lacunar infarcts, ht, dm, old age, fibrinogen, alcohol abuse, smoking, and female sex were the significant rfs. ht was the cause of ich in ( . %) and alcohol abuse ( , . %) and vascular anomalies ( , . %) followed. most alcoholassociated ichs occurred characteristically in the posterior fossa. frequencies of hospital admission of ich patients correlated positively with diurnal variation of temperature ( r = . , p < . ). aneurysmal rupture was the cause of sah in patients ( . %). three major sites of aneurysms identified on cerebral angiograms were the anterior communicating artery ( , . %), the middle cerebral artery ( , . %), and the posterior communicating artery ( , . %). thirty-nine patients ( . %) had no identifiable cause of sah. in korea curvilinear subinsular lesions have been noted on ct but the underlying pathology is unknown. we reviewed the cranial mr scans ( . tesla ge machine) of serial patients over the age of who had no cause for mr hyperintensities (hi) other than age or vascular risk factors. seven ( %) had linear subinsular h i (sihi). four of ( %) patients with sihi and of ( %) without sihi had marked periventricular h i compatible with subcortical arteriosclerotic encephalopathy (pvhi) ( p < . ). patients with sihi were older ( . yr) than patients without sihi ( . yr) ( p < . ). four of ( %) patients with sihi had hypertension program and abstracts, american neurological association or vascular risk factors. a further patient with diabetes, hypertension, and the opercular syndrome (bilateral facial, pharyngeal, and lingual weakness) had subinsular lesions on ct. the brain arteries were injected postmortem with a lead/ gelatin suspension, and mr of the whole brain and x-ray films of the brain slices were taken. bilateral sihi were seen on mr and corresponded with linear cavitary infarction pathologically, which on microangiograms was found to lie in the border-zone between cortical and basal penetrating arteries. we conclude that sihi are associated with older age and pvhi, and can be due to infarction in a deep watershed territory and can be associated with clinical deficits. hemodilution-treatment results in consecutive cases james l. frey, phoenix, az because precipitous neurological deterioration occurred during blood pressure reduction in a seminal case of lacunar infarction, subsequent patients with partial or evolving lacunar deficits were treated with hemodilution and blood pressure nonintervention to test the hypothesis that lacunar strokes represent perfusion failure. isovolemic hemodilution was performed using hetastarch with target hematocrit of to . results of pretreatment ct brain scans, carotid ultrasound, and echocardiograms were normal. nine patients recovered normal neurological function, and regained complete functional independence in close temporal correlation with hemodilution. mri brain scans demonstrated appropriate single white matter lesions in cases. no specific risk factor combination could be identified. n o patient has had recurrent stroke in follow-up from to months. response to hemodilution suggests a hemodynamic pathophysiology. successful treatment requires ( } blood pressure nonintervention and ( ) hemodilution prior to severe clinical deterioration. the hemodynamic classification for the carotid-cavernous sinus fistula (ccf) is important for the implication of prognosis and therapy, but satisfactory objective criteria for such differentiation is still lacking. retrospectively, we studied the application of extracranial duplex sonography in cases of ccf with emphasis on the hemodynamic parameters of resistivity index and flow volume. a correlation was made with the angiographic findings in an attempt to evolve an objective hemodynamic classification by this noninvasive method. the alterations in membrane metabolism and structure could be the primary etiological event in alzheimer's disease (ad) that results in the clinical and neuropathological findings. to investigate in vivo brain membrane phospholipid and highenergy phosphate metabolism in probable ad patients and control subjects, the building blocks (pme) and breakdown products (pde) of membranes and the high-energy phosphates pcr and atp were measured noninvasively by in vivo brain p mrs in probable ad patients ( males; females) and controls ( males; females). all subjects were assessed by mini-mental, mattis, and blessed scales. we found that, at clinical onset, ad females had elevated pme ( p = . ), decreased pcr ( p = . ), and decreased atp ( p = . ). similar changes were not seen in ad males at clinical onset, but the severely demented ad males had increased atp ( p = . ). correlation analysis for the ad patients revealed that increasing dementia was associated with decreasing pme ( p = . ; r = . ), increasing pde ( p = . ; r = . ), increasing pcr ( p = . ; r = . ), and increasing atp ( p = . ; y = . ). similar metaboliccognitive correlations were not seen in the controls. these results demonstrate alterations in membrane and energy metabolism at the earliest clinical stages of ad. increasing dementia correlates with markers of membrane degeneration and decreased utilization of high-energy phosphates. both of these findings suggest the dementia in ad is secondary to membrane changes resulting in synapse loss. alzheimer's disease: postmortem mri and histological correlates fen-lei f. chang, j. e. purisi, c. r. jack+ jr, and r. c. petersen, rochester, mn hippocampal atrophy, defined by mri-derived volumetric measurement, has been useful in differentiating alzheimer's disease (ad) patients from normals. since several studies have demonstrated anatomical and functional gradients along the rostral-caudal (r-c) axis of the hippocampus, it is tempting to speculate on the existence of a differential distribution of morphometric changes along this axis. the hippocampi from patients with clinically and pathologically confirmed ad were studied by postmortem mri and by reconstruction of serial histological sections. there was good correspondence between these two methods. the r-c length of the hippocampus in ad was preserved compared to normal. this length was longer on the left, both in normals and ad. the adassociated atrophy was due primarily to the reduction of the coronal cross-sectional area of the hippocampus. with increasing hippocampal atrophy, volume reduction was more prominent in the rostral area (pes hippocampus). this nonuniform reduction in volume may be associated with different connectivity patterns between rostral and caudal hippocampus. the purpose of this study was to determine the neuropathological validity of nincds-adrda criteria (nac) for probable and possible ad. mckhann et al ( ) provided clinical criteria for the categories probable ad and possible ad. the validity of these categories has not yet been reported. a retrospective, blinded evaluation of the complete neurological history, examination, neuroimaging, laboratory, and psychometric data was done for subjects from the state of florida brain bank. pathological classification, which was blind to clinical diagnoses, was in categories: pure ad, ad + , and other dementias. ninety-three percent of probable ad (n = ), whereas only % of possible ad (n = ) patients, had ad or a d + ( p = . ). pure ad was found in % of probable ad and % of possible ad patients ( p = . ). pathological evidence of coexisting parkinson's disease was present in % of all ad brains. these results suggest differential predictive power of nac for probable and possible ad, as suggested by the labels. nac are, therefore, usefui for research and clinical purposes. our prior study of falls in the elderly had shown significantly more white matter low attenuation (wmla) on ct scan among fallers than nonfallers, but no association between wmla and cognitive impairment among nondemented subjects. as these subjects were followed over time, it appeared that fallers were becoming demented at a more rapid rate than nonfallers. as a result, we analyzed the relationship between wmla and rate of change on the blessed test of information, memory, and concentration (bimc) in a combined cohort of subjects from the falls study and from a longitudinal study of dementia and normal aging. we selected of these subjects whose initial bimc score was less than (i.e., not already severely impaired) and who had at least yearly bimc evaluations. ct scans were scored on an -point ordinal scale for hemispheric wmla. linear regression was used to summarize the rate of change for each subjects' test scores. the rate of change on bimc was . points per year with standard error (se) of . among subjects who eventually became demented; nondemented subjects declined at . points per year with se of . . multiple regression analysis was performed with initial bimc score and wmla as the independent variables and rate of change of bimc as the dependent variable. wmla accounted for % of the variance (partial r = . , t = . , p < . ). these data suggest that elderly subjects with wmla may be at increased risk for rapid cognitive decline. such as g proteins and adenylate cyclase. the activities of adenylate cyclase, and of the g protein-associated enzyme activity, low-km glutamyl transpeptidase (gtpase), were assayed in membranes prepared from the postmortem brains of alzheimer-diseased and age-matched control subjects. both basal and fluoroaluminate-stimulated adenylate cyclase activities were significantly reduced in ad frontal cortex compared to control subjects ( p < . ; two-tailed student's t test). in addition, a significant, though smaller, reduction in basal gtpase activity also was detected in ad frontal cortex ( p < . ). in contrast, no significant change in the activity of either enzyme was detected in the hippocampus. the stimulation of gtpase activity by muscarinic and gababreceptor agonists was not altered significantly by the presence of alzheimer's disease. however, the degree of stimulation was much lower in human tissue compared to that observed using fresh rat brain, suggesting that the ability of receptors to activate g proteins declines post mortem. these results suggest that alzheimer's disease causes alterations in some key components involved in signal transduction. the association of lobar hemorrhage (lh) with cerebral amyloid angiopathy (caa) and that of caa with alzheimer's disease (ad) are well known. to determine how frequently lh and caa occur in ad, we reviewed patients with cerebral or cerebellar hemorrhage and caa. five patients were treated surgically, none was demented, died, and came to autopsy. eight patients died of lh and came to autopsy. of the autopsied patients, had ad, both clinically and neuropathologically. they comprised . % of cases of autopsy-confirmed ad in our laboratory. none of the other patients were known to be demented. five had senile plaques, with or without neurofibrillary tangles, in the hippocampus, and occasional senile plaques in the cortex, but none met the consortium for establishing a registry for alzheimer's disease neuropathological criteria for ad. ad patients were , , and years old and the ages of the nondemented patients ranged from to (mean = . yr). seven were younger than years of age. despite the frequent occurrence of caa in ad, we found that lh was uncommon. in addition, most of our patients with lh and caa were not demented and tended to be younger than ad patients with lh. these results indicate that the hf is involved primarily in acquisition or leaning processes and less so in retrieval of previously learned information. these findings relate to memory and structural brain changes found in normal aging. alzheimer's disease y . stern, l. stricks, g. alexander, . prohovnik, and there is an inverse relationship between parietotemporal cerebral blood flow and years of education in alzheimer's disease (ad) patients matched for clinical severity, which suggests delayed clinical manifestation of ad in patients with higher education (stern et al, soc neurosci abs ). we classified the lifetime primary occupations of ad patients using the dictionary of occupational titles of the us department of labor and derived factor scores describing intellectual, interpersonal, and physical job demands. after controlling for age, education, age at onset, illness duration, and dementia severity (mental status and activities of daily living), relative perfusion in the parietotemporal region (assessed using -xenon inhalation) showed significant correlations with job complexity (pl: r = -. , p < . ) and interpersonal (p : r = -. , p < . ) factor scores. in a stepwise multiple regression, job complexity and interpersonal skills increased explained parietotemporal flow variance by . % ( f = . , p < . ) over that explained by demo-graphic and severity indices; physical demands then accounted for another . % of the variance ( f = . , p < . ). we conclude that occupational demands, similar to but independent of education, may provide a reserve that delays the clinical expression of ad. richard mayeux and ming-xin tang, new york, n y risk factors for alzheimer's disease (ad) were collected from patients with ad and healthy elderly controls in an urban community population consisting of ethnic groups: black, hispanic, and white. advanced age (> yr) (or = . ; % ci . - . ) and head injury with loss of consciousness (or = . ; . - . ) were associated with ad, controlling for all known putative risk factors. factors such as low education (< yr) (or = . ; . - . ) and family history of ad (or = . ; . - . ) were not found to be significantly related to ad. head injury occurred in . % of the patients and .?% of controls. most ( %) head injuries in the patients with ad occurred after age , prior to disease onset. in controls with head injury, had experienced a head injury before age . the duration of unconsciousness was consistently longer in patients with ad than in the controls. the overall effect in each ethnic group was similar (or,, . ; . - . ). these results confirm and strengthen the previously described putative relationship between head injury and ad. we also conclude that both the severity and the timing of the head injury as well as the frequency of head injury in the population at risk may be important factors in understanding the causal relationship between head injury and ad. the purpose of this study was to determine how native language affects the cutoff scores in screening tests for dementia. there is a paucity of data o n this issue at present. screening tests used in the study were: folstein mini-mental state (mms); clockdrawing (clock); preparing a letter for mailing (mail); -item grocery list (list); and hamilton depression scale (ham). the subjects included ( demented) native english speakers (eng) and ( demented) native spanish speakers (spa) with memory complaints. diagnosis of dementia was determined by neurological, neuropsychological, and psychiatric evaluation. age and gender were unrelated to mms. in spa only, education was positively related to mms. ham scores were higher in de- and . (spa); c d and list did not add discriminative power to mms for eng but did so for list in spa. mms discriminates between demented and nondemented far better in english than in spanish speakers. only mail adds discriminative power to mms. a. heyman, g. fillenbaum, s. mirra, and participating cerad neuropathologists, durham, nc, and atlanta, ga the clinical diagnosis of alzheimer's disease (ad) has become more accurate in recent years due to the application of specific clinical criteria, wider use of neuroimaging procedures, and greater expertise among physicians. we report the frequency of clinical misdiagnosis of ad among patients who at autopsy were found to meet the rigorous clinical diagnostic criteria imposed by the consortium to establish a registry for alzheimer's disease (cerad) study. these patients included men and women (mean ages and yr, respectively) who were among the group of patients who died in cerad medical centers in the us between and . the clinical diagnosis of ad was neuropathologically confirmed in ( . %) of the cases. of these cases, varying degrees of concomitant cerebrovascular disease were present in % and coexisting parkinson's disease changes were found in %. in of the patients without neuropathological evidence of ad, the diagnoses were: lobar atrophy, diffuse lewy body disease, nonspecific neurodegenerative changes, and mesocorticolimbic dementia, respectively. the fifth patient showed no morphological abnormalities. on the basis of these results, it would appear that application of strict diagnostic criteria, as well as the use of brain scans and detailed clinical and neuropsychological tests by experienced clinicians, cannot yet distinguish some types of primary degenerative dementias from alzheimer's disease. we designed a scale that measures an underexplored facet of functional decline in alzheimer's disease (ad): the patient's dependence on others for supervising or performing activities. two hundred twenty-three informants for patients with mild ad (clinical dementia rating [cdr] = for ; cdr = for ) were interviewed and dependence was staged from to . interrater reliability was assessed by separate interviews of informants; agreement was % for dependence stage. dependence stage differed significantly at the cdr levels (chi square = . , p < . ) and correlated significantly with modified mini-mental state (mmms) ( r = -. , p < . ) and blessed dementia rating scale-part (bdrs) ( y = . , p < . ). seventy-eight percent of patients in a health-related facility were at stage or higher vs % of patients living at home. in a multiple regression model, both the bdrs and dependence scale accounted for unique portions of the variance in mmms, suggesting that they assess unique aspects of functional ability. dependence increased significantly in patients retested at year. we conclude that the dependence scale is reliable and relates to both disease severity and progression. formal assessment of dependence should prove useful for studies of the natural history of ad as well as for clinical trials. cortical-basal ganglionic degeneration (cbgd) is a disorder characterized by an asymmetrical akinetic-rigid syndrome and cortical signs such as apraxia, alien limb phenomena, and cortical sensory loss. dementia has been present in many cases, but always as a late manifestation. we report cases pathologically consistent with cbgd, presenting as primary degenerative dementia, fulfilling nincds-adrda criteria for probable alzheimer's disease (ad). the first patient presented with changes in memory and personality, language dysfunction, decreased verbal output, and shuffling gait. follow-up examinations over years showed progressive dementia, wide-based gait, and frequent falls. the second patient presented with complaints of memory loss. neuropsychological examinations showed progressive deficits in memory, attention, calculations, and visuospatial functioning. no movement disorder developed over years of follow-up. at autopsy, both patients had typical changes of cbgd and lacked pathological features of ad. definitive diagnosis of cbgd rests on both clinical and pathological criteria. cbgd should be considered in the differential diagnosis of patients with dementia resembling that found in ad, especially if extrapyramidal signs are present. a quick, easily administered and scored test for praxis is desirable in evaluation of neurological patients. during months in , each patient seen in the outpatient setting by the principal investigator (e. k.) received of praxis screening batteries. thirty-six patients were tested with a short battery. eleven had normal cognition based on neurological history, examination, and a short test of mental status. twenty-five had cognitive decline (cd). handedness, male/ female ratio, education, and mean age were similar in both groups. mean time of completion of the test was seconds in normals and * seconds in patients with cd. total scores (maximum ) were . * . in the normals and . in the cognitively declined patients. twenty-five other patients, with cd and with normal cognition, were tested with a longer battery containing oral/ facial, upper and lower limb, axial, sequential, and imitation subtests (maximum score ). normals completed the battery in i , and cognitively declined patients completed the battery in ? seconds. of the various subtests, tests of sequential praxis were performed most poorly by patients with cd: . * . in normals vs . * . in patients with cd. oral/facial praxis was least affected by cd: olivopontocerebellar atrophy (opca) is generally understood to be a nondementing neurodegenerative disorder affecting the cerebellum, lower brainstem, and spinal cord. one of us (s. k.) recently reported that postmortem cerebral cortex from patients with dominantly inherited opca shows a widespread reduction of cholinergic markers similar to that observed in alzheimer's disease (ad). we were interested to determine the status of other neurotransmitter systems in opca postmortem cerebral cortex. samples of frontal, parietal, temporal, and occipital cortex were dissected from confirmed cases of opca and age-matched controls. after processing, neuropeptide levels were measured by radioimmunoassay. concentrations of somatostatin were significantly reduced by to % in of the cortical areas of opca brain that were examined. the area that was spared was the inferior temporal gyrus, a region in which somatostatin levels are markedly reduced in ad. levels of neuropeptide y were normal in all areas, while concentrations of cholecystokinin, vasoactive intestinal polypeptide, and substance p were significantly increased in of the areas. these data show widespread neuropeptide changes in the cerebral cortex of opca postmortem brain. in contrast to cholinergic markers, the pattern of neuropeptide changes is different from what is observed in ad. it has been postulated that demise of the corticomotoneuron is the initial event in amyotrophic lateral sclerosis (als) and that the anterior horn cell dies as the result of antegrade glutamatergic excitotoxicity (muscle nerve ; : ). excitability of the corticomotoneuronal system can be tested by measuring threshold-to-cortical magnetic stimulation and the motor-evoked potential (mep)/compound muscle action potential (cmap) ratio, which estimates the number of corticornotoneurons stimulated. cortical threshold and mep/ cmap ratio were measured in patients early in the course of als. the mean time interval from onset of first symptoms was . months. mean threshold and mepicmap ratio measured . f . % and . rfr . %, respectively. in ( . %) patients, threshold was paradoxically low (< %, mean . +. . %) and in ( . %) patients there was no response. there was a significant ( r z = . ) inverse power relationship between cortical threshold and mep/cmap ratio given by . x mep/cmap-'j. six months later, / ( . %) patients still had low thresholds but the mean mep/ cmap ratio had dropped to . ? . % and in . % there was no response. we conclude that early in als the corticomotoneuronal pathways are abnormally excitable. this may explain early cramping and fasciculation, which characteristically diminishes as als progresses. one of the distinct clinical features in patients with amyotrophic lateral sclerosis (als) is loss of elasticity of skin. however, little is known concerning the biochemical nature of skin elastin in als. in our study, cross-links unique to elastin, desmosine and isodesmosine, were measured and compared in skin tissue (left upper arm) from patients with als and from age-matched controls. the contents of desmosine and isodesmosine were decreased significantly ( p < . and p < . , respectively) in patients with als (mean * sd, . ? . and . * . nmol/mg dry weight; range . - . and . - . nmol/mg dry weight, respectively) as compared with those of controls (mean sd, . * . and . . ;range . - . and . - . , respectively), and were negatively and significantly associated with duration of illness in patients with with amyotrophic lateral sclerosis als ( r = - . , p < . , and r = - . , p < . , respectively). the ratio of desmosine and isodesmosine was constant ( : ) in all samples analyzed. the decline in skin desmosine and isodesmosine is more rapid in als than in normal aging. thus, cross-linking of skin elastin is affected in als. (supported in part by n i h grants de , de , de , ar , ar , and nasa grant nag- - .) pl . natural history of amyotrophic ( ) collection of large numbers of twin pairs in disease of low prevalence is difficult. to circumvent this, we devised a new approach termed the "death discordant twin pair" method. eleven thousand deaths from motor neuron disease (mnd) were extracted from the office of population censuses and surveys during to . birth indexes from onward were searched for possible twins. for each twin so identified ( pairs), the national health service central registry located the relevant family practitioner committee and thence the co-twin's general practitioner. the search produced: ( ) living co-twins; ( ) embarked; ( ) dying as adults or infants; ( ) not mnd; and ( ) validity of the accuracy, sensitivity, and specificity of the world federation of neurology (wfn) subcommittee on motor neuron disease working group criteria for the clinical diagnosis of amyotrophic lateral sclerosis (als) has been tested against neuropathological criteria in autopsied patients (neurology, in press). integration of clinical and electrodiagnostic data to meet wfn criteria for possible, probable, and definite als was studied in this samegroup. patients received . ifr . (mean * standard deviation) electromyograms (emgs) per patient and included . . emg levels (bulbar, cervical, thoracic, lumbar) per patient. proportionately fewer emgs were performed as the level of diagnostic certainty at presentation increased: suspected ( . %), possible ( . %), probable ( . %), definite ( . %). in only . % of all patients studied did the first emg alone change the level of diagnostic certainty of the diagnosis of als. only . % of patients presenting with suspected als alone or with possible or probable als were associated with a change in level of diagnostic certainty following or more emgs. however, although increasing the number of emgs performed per patient may be associated with an increasing chance of increasing the level of diagnostic certainty ( emgdpatient = . %; emgdpatient = . %), selection of the level of emg analysis was more crucial. the "el escorial" criteria emphasize the importance of emg evidence of lower motor neuron involvement in a limb with clinical upper motor neuron signs. our analysis of emg studies in autopsy-confirmed als patients suggests that complete evaluation of bulbar and thoracic levels for lower motor neuron changes and complete evaluation of motor unit recruitment patterns are important for the integration of emg data with clinical data in the application of the "el escorial" criteria for the diagnosis of possible, probable, and definite als. sibships on guam annette grefe, john steele, linda flares, and stephen waving, birmingham, al, and umatac and mangikao, guam reports in the s indicated that % of guamanian chamorro patients with amyotrophic lateral sclerosis (als) gave october a positive family history. subsequent investigators have inferred that purely genetic factors are not responsible for guamanian als or its clinical variant, parkinsonism-dementia complex (pdc). we report the first chamorro sibships selected in an ongoing study of familial aggregations. the basis for the initial selection was that the youngest patient in the sibship had als. age of onset was to years (mean yr). fourteen of persons in these sibships were affected. others in the sibship developed pdc, progressive supranuclear palsy, or pure dementia later in life (mean yr, range - yr). these cases developed to years (mean yr) after onset of disease in the first sibling. the age at onset of the first case and the intervals between earliest and latest cases in such sibships may help to determine minimal and maximal latency (i.e., interval between exposure to an exogenous agent and onset of symptoms). our observations suggest that exposure may have occurred early (before age ) with varying and often long latency (up to yr). we also find that variability of clinical expression may be correlated with the age at onset. concentrating on the study of familial cases on guam may enhance the identification of the etiologic agent(s) of this prevalent and tragic disorder. the spinocerebellar ataxias are an uncommon group of genetic disorders that have been well characterized in north america and europe. information concerning these conditions in africa and other parts is scant. to address this problem, a large-scale survey has been undertaken in the cape province of south africa. in this investigation, more than persons in affected families have been appraised and investigated and phenotypic features have been analyzed in detail. linkage studies have been undertaken in families with similar phenotypes in which the condition was transmitted as an autosomal-dominant trait. human lymphocyte antigen (hla) typing was carried out on members of the families and linkage analysis was undertaken using the liped program to analyze the data with a correction factor for age of onset. maximum lod scores were: family a: . ( = . ); family b: . ( = . ); family c: . ( = . ); family d: . ( = . ). these results indicate linkage to hla in out of families. these findings provide support for the concept of genetic heterogeneity in these phenotypically homogeneous families. pcr typing with the reportedly more closely linked d s locus is now being undertaken in these south african families. h.-p. hartung, g. f. hoffmann, and g. becker, wunburg, heidelberg, germany recently, l- -hydroxyglutaric acidemia has been described as a novel metabolic disorder in children. we report the occurrence of this disease in adults. one of brothers developed at the age of an abnormal gait and dysarthria; in the other , clumsiness and walking delay were noted at age . symptoms progressed and at the time of admission, when the patients were and years, neurological examination revealed a spastic ataxic gait, limb ataxia, dysmetria, dysarthria, dystonic posturing, and mental retardation. ct and mr imaging revealed subcortical white matter changes with loss of arcuate fibers, folial atrophy, and leakage in the cerebellar vermis, as well as atrophic changes in the cerebellar hemi-acidemia program and abstracts, american neurological association spheres. on biochemical screening, highly elevated concentrations of l- -hydroxyglutaric acid were found in csf, plasma, and urine. the pathological accumulation of l- hydroxyglutaric acid in these adults, along with the clinical picture characterized by cerebellar, extrapyramidal, and pyramidal symptoms and oligophrenia, and the neuroradiological findings of severe loss of myelinated arcuate fibers in subcortical white matter, conform with what previously has been described in the few neuropediatric cases. the biochemical abnormality underlying accumulation of this organic acid remains elusive. this study was undertaken to differentiate primarily affected areas from functionally suppressed areas due to remote effect in aphasic patients with focal brain degeneration, using an activation method with - water, in comparison with [sf] -fluoro- -deoxy-~-g~ucose (fdg) positron emission tomographic examination at rest. the subjects were patients with slowly progressive aphasia showing contrasted clinical symptoms (nonfluent type vs fluent type). regional cerebral metabolic rate of glucose (cmrglu) was measured with intravenous injection of mbq of f- fdg at rest. regional cerebral blood flow (cbf) was measured with intravenous bolus injection of . gbq of - water in different conditions: at rest, under repetition tasks, and under naming tasks. changes of cbf were evaluated between a resting condition and task-performing conditions. regional cmrglu was decreased focally in both broca's and wernicke's areas similarly in these cases in spite of the difference in clinical symptoms, whereas the patterns of regional cbf changes were different. the fluent patient showed prominent activation in the bilateral frontal areas on repetition tasks. the nonfluent patient showed, whereas the fluent patient did not show, focal activation in the left occipitoparietal area on a naming task. evaluation with an activation method can provide detailed information about the pathophysiological process and the location of primary lesion. defective complex i activity has been linked to huntington's disease (hd) and parkinson's disease (pd). intrastriatal injection of inhibitors of complex i reproduces the pathological features of hd, and the neurotoxin mpp+ kills dopaminergic neurons by inhibiting complex i. defects in complex i have been reported in hd and pd, but the distribution of this enzyme in the brain is unknown. to map complex i in brain quantitatively, we developed an assay using e h)dihydrorotenone to label the enzyme in tissue sections. this high-affinity binding is saturable and is displaceable by rote-in brain none and mpp+. using pm rotenone to define nonspecific binding, more than % of binding is specific. highest levels of binding are found in kidney, followed by myocardium. moderate levels of complex i are seen in striated muscle and some brain regions. within the brain, binding varies more than -fold and is heaviest in the cerebellar molecular layer and dentate gyrus. lower levels of binding are found in cortex and striatum and very low levels are located in substantia nigra. this assay may help to clarify the role of complex i in neurodegenerative disorders. ( in , patients with medically intractable parkinson's disease underwent autologous adrenal medullary-to-caudate transplants at the university of california-los angeles (ucla). these persons had been followed for several years before operation at -or -month intervals. at each visit, their disability had been rated on the quantified ucla scale and the hoehn and yahr stage of disease. these provided a longitudinal assessment of the progression of disease in each patient, which could be compared to the rate of progression in the cohort of cases followed at ucla for years. in addition, the unified parkinson's disease rating scale provided supplementary data for the immediate preoperative and subsequent postoperative evaluations. the hours "off" also were recorded for the preoperative and postoperative periods. after operation, the same evaluations were performed by the neurologist who previously had cared for the patients. the longitudinal postoperative data revealed that at to months after operation, all patients improved. after years, continue to be less disabled than their preoperative baselines. the progression of their disease, while still evident, is nonetheless proceeding at a slower rate than before transplant. the fourth patient had brief improvement shortly after operation, but then rapidly worsened to his previous level. his disease has continued to progress at a rapid pace, unchanged from progression before operation. individuals at risk for huntington's disease h . p. h . kremer, w. shtybel, b. snow, c. clark, j . theilmann, m . r. hayden, and w. in huntington's disease (hd), caudate hypometabolism as demonstrated by positron emission tomography (pet) is a well-established feature in symptomatic patients. in individuals at risk, however, conflicting findings are reported. since we have performed pet scans in asymptomatic persons at risk for hd (age - yr) . linkage analysis with independent dna probes or subsequent evolution to clinical hd (in patients) allowed a risk estimate for subjects. twenty were considered to be at increased risk (? %), at decreased risk (< %), and in individual no modification could be given. nine subjects were not tested. a pet scan was considered abnormal if either the caudate/thalamus ratio or the caudate/whole-brain ratio of rcmrglu was more than these criteria, scans in individuals were abnormal. none had received a decreased risk. comparison of the ratios of increased risk, decreased risk, unmodified risk, and control subjects failed to show statistically significant differences (analysis of variance). follow-up of persons with an abnormal scan showed conversion to symptomatic status within years after the first abnormal scan. this result suggests that an abnormal pet scan in a person at risk for h d heralds the onset of choreic movements. robitaille, m . el-awar, b. clark, l. scbut, m. ball, l. young, r. currier, and k. sbannak, toronto, ontario, and montreal, quebec, canada; pittsburgh, pa, minneapolis, mn, portkmd, or, and jackson, ms we measured the levels of dopamine in striatum of patients with end-stage dominantly inherited olivopontocerebellar atrophy (opca). on average, dopamine levels were reduced in putamen ( - , as compared with controls), caudate ( - %), and nucleus accumbens ( - %). however, individual patient values showed a wide variation (normal to - ), indicating that striatal dopamine loss is a common, but not constant feature of opca. seven patients had marked putamen dopamine loss (- to - ) but without corresponding severe substantia nigra cell damage; this suggests a "dying-back'' phenomenon in which nerve terminal loss precedes cell-body degeneration. in this regard, opca may offer the possibility of examining nigrostriatal dopamine neuronal degeneration at an early stage. although patients were found to have severe nigral cell loss with near total ( - to - %) striatal dopamine loss, none had depression in parkinson's disease (pd) has been correlated with low cerebrospinal fluid (csf) levels of -hydroxyindoleacetic acid ( -hiaa). l-dopa may precipitate or exacerbate depression in % of pd patients. to determine if l-dopa affects -hydroxytryptamine ( ht) metabolism, patients were studied. four had pd. of these, were taking l-dopa and both were depressed. the diagnoses in the remaining were progressive supranuclear palsy (psp), striatonigral degeneration (snd), normal pressure hydrocephalus, and pseudoseizures with depression. neuropsychological examinations and lps of all patients were done. three patients were started on l-dopa (the previously untreated pd patients and the psp patient) and retested days later. one pd patient started on l-dopa became depressed. mood in the others was unchanged. csf was analyzed for ht and -hiaa. ht could not be detected in the csf of patients who were not taking l-dopa, but was easily detectable in all of the patients who were taking l-dopa. -hiaa levels were low in the untreated pd patients, and also in the patients with psp, snd, and pseudoseizures with depression. -hiaa levels were even lower in the l-dopa-treated patients. the ratio of -hiaa: ht (an index of ht turnover) was lowest in the l-dopa-treated pd patients who were depressed. low csf -hiaa in untreated pd may reflect depletion of brain ht. l-dopa may induce depression by inhibiting ht turnover in ht-depleted brain. p . ventroposterolateral medial pallidotomy in the e. fazzani, m. dogali, a. ben;, d. eidelberg, j. gianutsos, t. kay, b. newman, s. loftus, d. samehon, and l. laitinen, new york, n y , and stockholm, sweden in patients with parkinson's disease (pd), as a consequence of low dopamine there exists an increase in inhibitory output from the globus pallidus. ten patients ( men and women) with pd received unilateral ( right, left) ventroposterolateral medial globus pallidotomies (vplmp). the average patient age was years (range - yr), and the average duration of disease was years (range - yr). patients fluctuated between "on" chorea and "off" parkinsonism. hoehn and yahr stage "on" was i in , in ; and "off" was i in , iv in , and v in patients. unified pd rating scale (updrs) score averages hours off medicines ( hom) were activities of daily living (adl): and motor (mtr): preoperatively (preop). capit score averages preop hom were pronation-supination (ps): seconds (s), finger tap (ft): s, board (b): s for the most affected contralateral side, and gait: s ( patients could not walk). re-examination was done to days after pallidotomy. updrs scores hom decreased an average of %. three patients had major bilateral improvement in bradykinesia. rest tremor, prominent in patients, also was diminished. capit scores hom decreased to ps: s, ft: s, b: s; the average gait of the patients who could walk preop improved to s. there were no side effects. vplmp leads to an immediate overall significant improvement in patients with pd. s. kish, y . there is a growing interest in the genetic aspects of parkinson's disease and other basal ganglia disorders. we have studied families whose ancestors immigrated to north america from contiguous regions of northern germany and southern denmark. the pedigrees contain , , and individuals spanning , , and generations with , , and affected members, respectively. autosomal-dominant inheritance is clearly present in families and probable in the third. typical levodopa-responsive parkinsonism with bradykinesia, rigidity, resting tremor, and impaired postural reflexes uniformly develops in affected individuals from all families. n o downgaze impairment, pyramidal signs, sensory disturbances, cerebellar dysfunction, or orthostatic blood pressure changes have been observed. dementia, however, has developed in a few elderly individuals, especially in family. laboratory studies are normal. mri shows moderately enlarged ventricles and cortical atrophy. -fd positron emission tomography demonstrated reduced striatal uptake in examined patient and normal uptake in l individual at risk. autopsy of only subject has been performed (in ). brain weight was , grams and there were no obvious gross abnormali- program and abstracts, american neurological association tuesday, october ties, but microscopic examination was limited. further research on these families is planned. electrophysiological study s. maurri, m . cincotta, a. ragazzoni, g. descisciolo, and f. barontini, florence, ltah many neurophysiological examinations were conducted of a -year-old woman with familial mirror movements. n o other neurological abnormalities were detected. examination included voluntary electromyographic (emg) activity from various muscles, f-wave as well as short-and long-latency reflex responses of the thenar muscles from electrical stimulation of the median nerve, mapping of motor-evoked potentials (meps) to transcranial magnetic stimulation, movement-related cortical potentials (mrcps), and somatosensory-evoked potentials (seps). emg documented mirror activity in the upper limbs, most marked in the muscles of both hands. onset latency of emg activity in response to an auditory stimulus was identical in active and mirror muscle. long-latency responses from median nerve stimulation were recorded on contralateral as well as ipsilateral thenar muscles. short-latency reflexes and f wave were strictly ipsilateral. unilateral scalp magnetic stimulation evoked bilateral responses at similar latencies in the thenar muscles; midline scalp stimulation activated no responses. scalp distribution of median nerve seps and of mrcps associated with self-paced thumb abduction were normal. our findings suggest that congenital inherited mirror movements in otherwise normal subjects can be generated by corticospinal fibers pro jecting to ipsilateral motoneurons of the spinal cord. we have previously identified dysphagia and constipation (both slow transit and defecatory dysfunction types) as common gastrointestinal (gi) problems in parkinson's disease (pd). since apomorphine has been shown to be capable of terminating off-periods when injected subcutaneously, we have evaluated the effects of apomorphine injection on objective parameters of dysphagia and bowel dysfunction in pd patients. nine subjects underwent the following battery of studies to characterize their pd features, swallowing, and bowel function: gi assessment survey, unified pd rating scale, videoesophagram, colon transit study, defecography, and anorectal manometry. specific abnormalities on the studies were noted and the most abnormal study was repeated after subcutaneous administration of mg apomorphine. all individuals were pretreated with domperidone mg four times daily for days prior to apomorphine administration. improvement in both esophageal motility and deglutition was noted in the individual in whom videoesophagram was repeated. for the other patients, defecography or anorectal manometry was performed. significant improvement in specific parameters was demonstrated after apomorphine administration, but individuals experienced syncope during radiographic procedures. we conclude that subcutaneous apomorphine administration holds promise as a potential therapeutic approach to dysphagia and, especially, bowel dysfunction in pd, but that further investigation and refinement are necessary. asymmetrical effect of unilateral thalamotomy or subthalamotomy on tremor in parkinson's disease nico diedericb, christopber g. goetz, glenn t . stebbins, harold l. klawans, k. nittner, a. kozrlosakis, p. sanker, and v . strum, cologne, germany, and chicago, il in the past, stereotactic operation was a regular treatment for unilateral tremor in parkinson's disease (pd). however, follow-up studies were usually short term and always unblinded. we examined pd patients in long-term follow-up (mean . yr after operation) who underwent unilateral thalamotomy for parkinsonian tremor. we used videotapes and the unified parkinson's disease rating scale to blindly compare tremor ipsilateral and contralateral to the side of operation. since the patients were specifically selected for stereotactic operation because of asymmetric tremor, we reasoned that a sign of long-term efficacy would be current postoperative reversal of tremor side predominance. upper extremity tremor was significantly better contralateral to the side of operation compared to the ipsilateral side ( z = . ; p < . ). for the lower extremities the difference was not statistically significant. in chronic follow-up, stereotactic operation improved the absolute magnitude of arm tremor or ameliorated its rate of progression. since asymmetric bradykinesia and dyskinesia were not prerequisites for the choice of surgical side, we cannot make any conclusion about longterm impact of operation on these features. subtle extrapyramidal signs resembles that of patients with parkinson's disease m . richards, k. marder, l. cote, y . stern, and r. mayeux, new york, n y to investigate the relationship between extrapyramidal signs (eps) and cognition, eps severity and neuropsychological function were assessed in normal elderly individuals and nondemented patients with idiopathic parkinson's disease (pd) from a community-dwelling cohort in new york city. multivariate analysis of variance (manova) indicated poorer neuropsychological performance ( p = . ) in pd patients on verbal memory, orientation, verbal fluency, visuomotor construction, and psychomotor speed, but not naming, abstract reasoning, or matching. controlling for eps severity abolished these differences. one hundred fourteen ( %) of the normal individuals had subtle eps (mostly postural abnormality, bradykinesia, or rigidity) but no identifiable neurological disorder. manova indicated poorer neuropsychological test performance ( j = , ) in these individuals than in normals without eps on verbal memory, orientation, abstract reasoning, naming, verbal fluency, matching, and psychomotor speed but not visuomotor construction. we conclude that: ( ) cognitive impairment in pd is specifically associated with eps, and ( ) a similar association occurs in individuals with subtle eps but no neurological disorder. whether this represents a preclinical stage of pd or ad is yet to be determined. disease in olmsted county, minnesota emre kokmen, fatma sibel ozekmekci, c. mary beard, and peter c. o'brien, rochester, m n , and istanbd, turkey there have been many studies of prevalence of huntington's disease (hd) in diverse populations around the world. to study the incidence, we took advantage of the availability of detailed health care records for the population of olmsted county, mn, from mayo clinic, its affiliated hospitals, olmsted medical group, county hospital, state hospital, records of solo practitioners, nursing homes, death certificates, and autopsy records. we reviewed all records with a diagnosis of hd, huntington's chorea, chorea major, and chorea otherwise unidentified, and sought evidence for progressive chorea, progressive cognitive and/or behavioral dysfunction, and family history compatible with autosomal-dominant inheritance with onset of symptoms in the period between january , , and december , , while the patient lived in the geographic boundaries of olmsted county. we found males and females who met these criteria. average annual incidence rate (age/sex adjusted to us white population) for hd for this -year period was . cases/ year/ , population. we also estimated prevalence by taking account of in-migration, out-migration, and deaths. the agelsex adjusted ( ) prevalence for - - was . , and for - - it was . / , . the small number of cases caused the instability of the prevalence rates, but our rates are similar to rates reported in other populations. alton e . btyant, , l. breeden hollis, john a. hamjian, john d. wooten, ill, and francis . walker, nc we described patients with unusual episodic movement disorders and normal diagnostic work-ups: a -year-old woman who had recurrent episodes of tonic jaw deviation and forced right-eye closure; a -year-old woman who developed unexplained pain and subsequent spells of tonic inversion of the left leg; a -year-old man who presented with a bizarre episodic right-arm tremor; and a -year-old woman who experienced intermittent abdominal undulations. examination of the affected body part provoked or enhanced symptoms in all patients. using suggestion and placebo activation in the form of a medicated patch, intravenous saline, or cervical massage, we first induced and then aborted typical episodes of their abnormal movements. postinduction discussions of the procedure led to a marked reduction in the frequency of attacks in patients. activation procedures are useful in diagnosing psychogenic disorders because they demonstrate that situational, not medical, factors govern the expression of the abnormal behavior. we speculate that patients who are refractory to simple suggestion may respond to induction because it offers the potential of validating their symptoms. as in the case of psychogenic respiratory distress or pseudoseizures, positive induction can assist in counseling and symptom control. had alzheimer's disease with parkinsonism (adp), had essential tremor (et), had cerebellar tremor in multiple sclerosis (ms), and had tardive dyskinesia (td). clozapine was used either to treat psychosis ( pd, adp, dys, td) or tremor ( pd, et, ms). two pd patients were retrospective analysis of patients counted twice, who was treated for psychosis and then tremor and who was treated on separate occasions for psychosis with different responses. all dys patients improved, with complete resolution of their dystonia on changing antipsychotic drugs. the patients with et ( mg) and ms ( mg) improved mildly but sedation and clumsiness caused drug discontinuation in the ms patient. one adp patient ( . mg) responded well and the other became sedated and confused ( mg). the p d responses for psychosis at a dose range of . to mg daily were good ( ), very good ( ), and excellent ( ), whereas were intolerant. pd tremor responses were good ( , very good ( ), excellent ( ), and poor ( ) at doses of . to mg daily. one patient died of unrelated causes shortly after initiation of the drug. adverse effects included sedation, weight gain, hypersalivation, fainting, clumsiness, transient granulocytopenia, and "spasms" necessitating discontinuation in patients ( pd, l td, and l ms). tourette's syndrome and attention-deficit disorder patients s. m. silverstein, p. g. coma, d. palumbo, l. west, and r. kurlan, rochester, n y impaired attention is a common comorbid behavioral feature of tourette's syndrome (ts) and a key clinical feature of attention-deficit hyperactivity disorder (adhd). however, the pattern of attentional impairments reported in adhd has not been observed in ts. we therefore compared ts patients ( male, female; mean age ? yr), adhd patients ( male, female; * yr), and normal controls ( male, female; ? yr) on specific neuropsychological (np) and computer-administered tasks of attentional ability. adhd, but not ts, subjects performed significantly worse than controls on the n p tasks (digit symbol, perceptual speed) and had a trend toward poorer performance on a computerized measure of attention. however, both the adhd and ts groups had significantly greater test performance variability on some, but not all, tasks and had more subjects with deviant scores. among ts patients, higher scores on an obsessive-compulsive disorder (ocd) inventory and a greater number of adhd symptoms correlated significantly with poorer performance on the attentional tasks. moreover, ts patients with observed tics during testing had greater attentional impairment than those without tics. these results suggest that: ( ) many adult ts patients do not have impaired attention; ( ) attentional impairment in ts differs from that observed in adhd; and ( ) attentional impairment in ts is associated with the full neurobehavioral spectrum of ts (i.e., tics, ocd, and adhd). frank r. sharp, cathleen miller, thomas rando, and steven greenberg, san francisco and palo alto, c a six patients are described with choreoathetoid movements and marked proprioceptive sensory loss. one patient had a traumatic injury to the right parietal cortex that produced severe proprioceptive sensory loss and choreoathetosis in the left arm. another patient had a left thalamic infarction that resulted in profound proprioceptive sensory loss and chorea on the right side of the body. two patients had cervical spinal cord disease, proprioceptive sensory loss, and diffuse choreoathetosis. another patient had dorsal root ganglionitis associ-a hypothesis program and abstracts, american neurological association ated with small-cell lung carcinoma that produced diffuse loss of all sensory modalities and chorea. the last patient had an ulnar sensory neuropathy and choreic movements of the fifth finger. lesions anywhere along the pathway that transmits limb proprioception may cause pseudochoreoathetosis. furthermore, choreoathetosis without sensory loss caused by focal lesions of striatum may occur because of disruption of cortical proprioceptive inputs to striatum-perhaps explaining why most focal lesions of striatum do not produce chorea. sporadic inclusion-body myositis (s-ibm) and autosomalrecessive hereditary inclusion-bod y myositis (h-ibm) are of unknown cause and pathogenesis. in both there are muscle fibers with rimmed vacuoles containing to -nm cytoplasmic tubulofilaments (ctfs) and denervation atrophy; in s-ibm, but not h-ibm, there is a varying degree of inflammation. vacuolated fibers contain ubiquitinated inclusions (askanas ) and congo-red positivity indicating amyloid (mendell ). because immunoreactive p-amyloid precursor protein (app) and p-amyloid protein (p-ap) are constituents of ubiquitinated senile plaques in alzheimer's disease (ad) brain, we studied immunolocalization of app and p-ap fibers in ibm muscle using antibodies against: ( ) non-p-ap fragments of app, viz. (a) c-terminus (residue - courtesy d. selkoe) and (b) n-terminus (residue - courtesy b. frangione and d. levartovsky); ( ) p-ap (sequence - , courtesy g. glenner, and sequence - courtesy d. selkoe); ( ) ub (chemicon). in of ibm patients, including one h-ibm, % of the vacuolated muscle fibers contained large or several small app and p-ap immunoreactive (ir) inclusions, which by double-labeling fluorescence were closely colocalized with each other and with ub-ir. none of control muscle biopsy specimens (including polymyositis) contained app-ir, p-ap-ir, or ub-ir inclusions characteristic of ibm. control experiments utilizing omitted, replaced, or absorbed primary antisera were negative. p-ap, a product of proteolytic cleavage of app, is receiving attention regarding the pathogenesis of ad. our study provides ( ) the first demonstration of app and p-ap accumulations in abnormal human muscle, and ( ) raises the possibility that in ibm muscle and ad brain rhey may form from similar cellular events. jeffrrey d. rothstein, lin jin, and ralph kuncl, baltimore, m d the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (als) is unknown. accumulating evidence suggests that the disease is characterized neurochemically by a derangement in the control of neurotransmitter glutamate metabolism: csf levels of glutamate and aspartate are ele-of motor neuron degeneration vated and their high-affinity transporter is defective in brain and spinal cord. inefficient glutamate transport, and subsequent chronic increase in extracellular glutamate, could be responsible for selective motor neuron death. to test the hypothesis that chronic defects in glutamate uptake can produce motor neuron toxicity, we developed a tissue culture model employing organotypic rat spinal cord maintained under conditions of chronic glutamate uptake inhibition. slices ( pm) of lumbar spinal cord from -to -day-old rat pups were cultured on millicell membranes. chronic uptake inhibition was produced by culturing tissue in the presence of threohydroxyaspartate (tha) or pyrrolidine-dicarboxylic acid, both known to be specific inhibitors of glutamate transport. tha produced chronic elevation of glutamate in the medium and produced motor neuron toxicity after to days in culture using pm tha, and after days using pm tha, as determined by assay of tissue choline acetyltransferase (chat) activity and by histological analysis of -micron plastic sections. motor neuron toxicity was completely blocked by the non-n-methybaspartate (nmda) antagonists cnqx or nbqx, but not by the nmda antagonist mk- . this model demonstrates that the chronic loss of glutamate transport in als can produce motor neuron degeneration and that motor neurons appear to be susceptible to non-nmda-mediated glutamate toxicity. guillain last year, we described a distinct acute paralytic syndrome in children and young adults from northern china and differentiated it from guillain-barre syndrome (gbs) by epidemiological, clinical, and nerve conduction (nc) features. to distinguish chinese paralytic syndrome (cps) from gbs more clearly, we measured nc in cps patients (mean yr, range . - yr) and in gbs patients from johns hopkins (mean yr, range - yr). sensory nc was normal in all (n = ) but nerve of cps patients, whereas sensory nc was frequently abnormal in gbs patients: median nerve, %; ulnar nerve, %; and sural nerve, %. motor n c also differed between the groups. in all nerves, distal latency (dl) was significantly longer in gbs than in cps. for example, in the median nerve, mean dl was . ms (se ) in gbs and . ms ( . ) in cps ( p < . ). motor conduction velocity was significantly reduced in gbs median and ulnar nerves compared with cps nerves. f-wave latency was significantly longer in gbs median nerves than in cps nerves. these data support the distinction both clinically and electrodiagnostically between cps and north american gbs. the use of n c may be especially important in field epidemiological studies in separating the disorders. clinical manifestations and gene analysis of the first japanese kindred yoshihide sunada, teruo shimizu, lchiro kanazawa, and toru mannen, tokyo, japan familial amyloidotic polyneuropathy type iv (fap iv) has been clustered in the finnish population and only a few cases have been reported from the netherlands. denmark, and united states. we describe the first japanese family with fap iv. the family originates from nagano prefecture, a mountainous district in the middle part of japan, and has no relationship to the finnish population. this family has members in generations, and individuals are affected with slowly progressive cranial neuropathy and corneal lattice dystrophy. the genetic trait is autosomal-dominant. polarizing microscopy and immunohistochemistry show abundant amyloid deposits reactive to an anti-gelsolin monoclonal antibody. direct sequence analysis of a dna fragment spanning codon of the plasmagelsolin cdna from the propositus, and restriction analysis using a modified pcr from other family members demonstrate a single base substitution, g to a at the first base of codon , which is identical to the mutation of finnish fap iv. this suggests that the mutation causes the fap iv phenotype regardless of ethnic background. gene expression focal puncture injury has been used as a model to study degenerative and regenerative responses of skeletal muscle. previous studies have demonstrated the ultrastructural and metabolic effects of muscle injury. however, the early genomic response to focal injury is presently unknown. we asked whether the immediate early genes (iegs) or early response genes-~$ , c-jun, nur , and junb-are responsive to muscle injury. these iegs encode transcription factors and are expressed rapidly after cell-surface stimulation. we have previously shown that surgical denervation and neural stimulation of muscle induced differential patterns of ieg expression. in this study, we produced injury of mouse gastrocnemius muscle by injection of c . of normal saline. we used the contralateral (uninjected) muscle as a control and examined the milna levels of each of these iegs. we found that ~$ and junb levels were increased at and hours and returned to basal levels by hours. in contrast, mrna levels of nur and cjun remained unchanged. this pattern of ieg response is distinct from that seen after muscle stimulation or denervation. the selectivity of this pattern suggests that ieg expression may play a role in the response of muscle to injury. amyotrophic lateral sclerosis (als) is a degenerative disease that leads to the restricted loss of motor neurons (mn). the reason for the selective death of mn remains unknown. we hypothesize that mn-enriched or mn-specific genes are important for normal m n function and that their disturbance may play a role in the pathogenesis of als. we have produced clonal hybrid cells derived from embryonic and neonatal spinal cord m n for the study of m n gene properties. some of these hybrid mn clones express traits typical of mn, such as high levels of choline acetyltransferase enzyme activity and message, glycine receptor message, and neurofilament and neural cell adhesion molecule proteins. we are using molecular techniques to identify novel mn-enriched or mn-specific genes in these cells. with this strategy, we have identified several cdna clones preferentially expressed in mn hybrid cells but not in the parental neuroblastoma cells by differential hybridization of an embryonic mn hybrid cdna phage library. we are extending these observations by performing subtraction hybridization experiments. these results suggest that mn-enriched or mn-specific genes can be identified, and may lead to a greater understanding of the etiology of als. there is a syndrome of slowly progressive, mid-adult-onset fasciculating progressive muscular atrophy (pma) affecting upper more than lower limbs, without bulbar or corticospinal signs, more often in males, associated with igm monoclonal gammopathy, and no nerve conduction block. two such men, ages (a) years and (b) years, duration and and one-half years, csf protein and , had failed to achieve sustained improvement with: prednisone, cyclophosphamide, total-body irradiation, and multiple lymphoplasmaphereses in a; and interferon alpha a in b. intravenous immunoglobulin (ivig), . gm/kg/day, has provided dramatic benefit, sustained and increasing for > and > months to date. (there is a continuing base of depotestosterone, mg weekly, which initially alone provided very minimal improvement.) strength increase was evident at and days after the first course of daily ivig infusions. it further increased for to and one-half weeks after treatment, and then began to diminish. repeat -day treatment weeks after the first course resulted in summated improvement, now sustained and enhanced by an average of treatment per week. quantitated strength testing by a blinded observer has shown a -fold to >loo-fold gradually increasing muscle function in all limbs. patient a regained the ability to feed himself, get out of a chair, walk unaided, and go up steps; quantitated hip flexors increased -and -fold. patient b regained the ability to feed himself, take care of personal toilet needs, walk securely, and drive miles; quantitated hip flexors increased -fold, and biceps flexions increased from with no weight to > reps while holding -pound weights. bukhara jews: a new cluster with typical oculopharyngeal muscular dystrophy (opmd) is a rare, late-onset myopathy with autosomal-dominant inheritance. its ultrastructural hallmark is the finding in muscle fibers of intranuclear tubular filaments of . -nm outer diameter. most opmd cases were described among french canadians; in france, the homeland of their ancestors, the prevalence is / , (brunet et al, ) . in israel's central area live approximately , jews who have immigrated from the bukhara and samarkand regions in uzbekistan. they represent a homogeneous ethnic group with its own language and community life. among them we have identified opmd in families ( affected individuals). the inheritance, clinical, electrophysiological, and histological features of these pa-tients are similar to those described in other pdts of the world, with typical intranuclear inclusions seen on electron microscopy. the minimal estimated prevalence of opmd in this population is approximately : . this cluster of opmd among bukhara jews is the second largest in the world. because many bukharian families are large, they may be suitable for linkage genetic studies. human muscle during ontogenesis e . scarpini, g. conti, p. l. baron, and g. myoblast transfer has been proposed recently as a possible therapy for duchenne muscular dystrophy patients. because immune rejection can represent a major problem in myoblast implantation, immunological characteristics of human muscle should be investigated. previous studies showed that human muscle cells cultured in vitro can constitutively express human lymphocyte antigen (hla) class i, but not hla class . furthermore, human y-interferon induces the surface expression of hla class i on mononuclear myoblasts, but not on multinucleated myotubes. however, whether the cells produce and present the antigen by themselves or take this material from the environment, where it could be released by infiltrative cells, is not yet clear. in this study, we analyzed hla molecules at the protein level by immunocytochemistry with monoclonal antibodies against different hla-dr epitopes and hla-abc molecules on frozen serial sections of human muscle during development and at the adult stage. human muscle infiltration by macrophages and monocytesmacrophages also were studied with m and leum specific monoclonal antibodies at the same stages of development. our results show that during muscle development and maturation, hla-dr and hla-abc antibodies do not label muscle fibers but some m -and leum -positive cells within the muscle. these data can be useful to understand the role of infiltrating monocytes-macrophages in the muscle immune response. mounting evidence suggests that excitotoxicity, mediated via the glutamate receptor, is involved in the pathogenesis of amyotrophic lateral sclerosis (als), as well as in other neurological diseases. we therefore initiated an open label, phasen i trial of highdose dextromethorphan (dm), a noncompetitive, selective n-methybaspartate antagonist, in als. patients began with mg/kg/day, divided into doses, and incrementally escalated their medication to mg/kg/day or their maximum tolerable dose. thirteen patients, all extensive metabolizers of dm, were enrolled. total daily doses ranged from . to mg/kg. major side effects were lightheadedness ( ), slurred speech ( ), and fatigue ( ). no biochemical, hematological, or neuropsychiatric abnormalities occurred after up to months of maximal therapy, except for depression in patient. plasma kinetics of dextrorphan (dt) (the major metabolite of dm) were studied after an acute oral dose of . mg/kg dm. median elimination halflife was . hours. plasma dt concentration peaked at a median of hours, with a median cmax of . pm. median amyotrophic lateral sclerosis cerebrospinal fluid/plasma dt ratio was . . this study demonstrates the feasibility of long-term, high-dose dm therapy. we are now conducting a phase i study of highdose dm in als, designed to assess its efficacy. polyneuropathy: a chronic inflammatory demyelinating polyradiculoneuropathy variant? d. cros, k. h. chiappa, s. patel, and s. gominak, boston, ma, we describe patients ( men, woman) with a pure, adultonset sensory neuropathy. the course was chronic in all cases. three patients had a relapsing-remitting course over to years with several attacks every year; the onset was gradual and followed by a plateau in the fourth patient. all patients had positive and negative sensory symptoms, and had positive motor symptoms (fasciculations). in all patients, muscle power was normal at the time of peak deficit. all were areflexic and had large fiber sensory deficits, and patients had sensory ataxia. three patients had elevated csf protein, whereas the csf was normal in patient. mri demonstrated marked thickening of the lumbosacral spinal roots in patient. motor conduction studies were normal in all patients, and mild f-response abnormalities were noted in . neurophysiological investigations of the sensory pathways were abnormal in all. three patients had several studies over a -year period. sensory nerve action potentials were unobtainable in patients, and normal in the others. median and tibial somatosensory-evoked potentials showed conduction slowing consistent with demyelinating lesions affecting the peripheral sensory pathways, either globally or focally in the proximal segments. two patients appeared to respond to plasma exchange or intravenous immunoglobulin therapy, or both. glial fibrillary acidic protein cells in experimental motoneuron disease raul n . mandler, pam c. allgood, and james a. wallace, albuquerque, nm neuronal degeneration in human and animal motoneuron disease has been emphasized, but glial phenotype alterations have not been studied as extensively. we carried out a developmental and topographic study of astrocyte expression in the wobbler mouse model of motoneuron disease. wobbler mice and normal littermates were studied at , , , and weeks of postnatal development. anesthetized animals were perfused intracardially with paraformaldehyde. spinal cords were dissected and landmarks were identified carefully for systematic study. sections were stained with monoclonal antibodies against glial fibrillary acidic protein (gfap) neurofilament and neuron-specific enolase. cell quantitation was done with video-enhancing microscopy. in symptomatic animals, marked increases in gfap staining were found in rostral and caudal spinal cord areas. quantitation studies revealed a to -fold increase in gfap+ cells in the wobbler. we conclude that gfap+ cells are markedly increased in the wobbler mouse at cervical, thoracic, and lumbar areas. this cell may also be relevant in motoneuron disease pathogenesis. ( indirect evidence suggests that polio virus may persist in the human cns years after initial infection and may be a cause for the post-polio syndrome. to evaluate whether the polio virus genome can be detected in the cns of patients with previous polio infection, we identified patients who had died with autopsy findings and clinical history consistent with poliomyelitis. rna was extracted from paraffin-embedded sections of brain or spinal cord and subjected to reverse transcription followed by dna amplification by polymerase chain reaction (rt-pcr) using primers specific for heat shock protein (hsp ) and a conserved region of the polio viruses. hsp mrna could be detected in all specimens, indicating that amplifiable rna had been isolated. in no specimens could polio virus rna be detected. this study suggests that polio virus does not persist in the human cns in quantities detectable by the sensitive pcr method. with cmt type , seen at mayo clinic rochester between and , for the frequency of selective calf weakness in cmt type , the form of cmt most similar clinically to distal sma. anterior compartment weakness exceeded calf weakness in patients ( %); anterior and posterior involvement was equal in ( %). calfweakness exceeded anterior compartment weakness in patient ( %). selective calf weakness in distal sma thus helps distinguish this disorder from cmt type , and similarly from distal sma with weakness resembling cmt, in that we are unaware of the distributions in distal sma occurring in the same family. given the possibility of genetic heterogeneity, linkage studies of distal sma probably should include patient selection criteria such that the distribution of leg muscle weakness is homogeneous. p . conjugal amyotrophic lateral sclerosis: amyotrophic lateral sclerosis (als) is a sporadic neurodegenerative disorder of unknown cause. unusual cases may provide etiologic clues. we report a married couple, both of clue to etiology? whom developed als in year. the couple grew up in southeastern pennsylvania and attended the same schools. they married after high school and have healthy children. in september , a -year-old woman noted right-hand weakness and associated fasciculations that progressed to the entire right upper extremity. by january , the lower extremities were asymmetrically weak and fasciculating. she then developed left-arm weakness, d ysarthria, dysphagia, and emotional incontinence. she had hyperreflexia and bilateral extensor plantar responses. then, in may , her husband, aged , noted difficulty whistling, which progressed to frank dysarthria. later, he developed dysphagia, emotional incontinence, and weakness, wasting, and fasciculations in the upper extremities. hyperactive gag, jaw, and limb reflexes were present. in both, electrodiagnostic testing revealed widespread evidence of lower motor neuron degeneration. numerous laboratory tests were normal. although these cases may represent a chance association, the development of als in a young husband and wife suggests a possible environmental cause. the authors welcome suggestions about these cases from the neurological community. conduction block in demyelinating neuropathies usually is assessed from differences in the sizes of surface-recorded maximum m-potentials evoked by supramaximal stimulation at successively more proximal sites along the course of motor nerves. as the maximum m-potential is comprised of many bitriphasic surface-recorded motor unit action potentials (muaps), differences in the relative latencies between muaps may lead to phase cancellations, reducing the m-potential size and rendering any quantitative assessment of the extent of conduction block relative to phase cancellation difficult. cooling a muscle (not the nerve), however, by as much as °c increases the negative peak durations of muaps by as much as to times and moves the point at which maximum phase cancellation might occur to some theoretical point well proximal to the spinal roots. in cases of guillain-barre syndrome (gbs) studied to date, cooling produced little change in percent reductions in m-potential negative peak areas between successively more proximal sites of stimulation. this finding suggests that "true" conduction block rather than interpotential phase cancellation best explains reductions in m-potential size at successively more proximal sites of stimulation in gbs. associated with trimethoprim-sul famethoxazole rare cases of primarily motor polyneuropathy have been associated with the use of sulfonamides. the incidence of polyneuropathy has diminished substantially with the abandonment of earlier methylated compounds. we describe patients who developed allergic phenomena, including a skin rash and debilitating, painful sensory and autonomic polyneuropathy within days of receiving trimethoprimsulfamethoxazole. in patient, examination revealed resting tachycardia, marked blood pressure orthostasis and near-program and abstracts, american neurological association syncope, hyporeffexia, urinary incontinence, and reduced sensation distally in the lower extremities. his cerebrospinal fluid was acellular with a protein of mg/dl. the other patient showed a resting tachycardia, sluggish pupils, reduced distal vibration perception, and hyperpathia of hands and feet. conventional nerve conduction studies demonstrated normal motor results in both patients, absent or reduced sensory amplitudes in the first patient, and normal sensory results in the second. autonomic studies identified profound abnormalities in testing of sympathetic skin potentials, sinus arrhythmia, and valsalva's ratio. in both cases, nerve biopsy was not performed for fear of exacerbating the patient's hyperpathia. subsequent hemodynamic and electrophysiological testing showed improvement in autonomic function, paralleling the patients' clinical amelioration. although uncommon, a painful, sensory and autonomic, partially reversible polyneuropathy may develop after the use of trimethoprim-sulfamethoxazole. the remote effects of botulinum a toxin injections into vocalis muscles for treatment of focal laryngeal dystonia were investigated using single-fiber electromyography (sfemg). botulinum a toxin injections have been proven effective therapy for various dystonic disorders including focal laryngeal dystonia, blepharospasm, and torticollis. previous sfemg studies have demonstrated remote effects of the toxin in noninjected muscles after treatment for both blepharospasm and torticollis. these effects include an increase in fiber density, mean jitter (mcd), and percentage of fiber pairs with increased jitter. other researchers have postulated that the distant effects of this toxin may be related in part to the dose of botulinum toxin injected. to investigate this hypothesis we have studied patients treated for focal laryngeal dystonia because the amounts of toxin required are / th to / ooth of the doses used to treat other dystonias. using electromyographic (emg) guidance, bilateral injections of . or . mouse units of botulinum a toxin were injected into each vocalis muscle of patients. each patient had significant improvement in phonotory function within hours after injections and have been followed serially (usually within weeks and again at mo) after injections, with sfemg recordings of the left extensor digitorum communis and sternocleidomastoid muscles. five patients have had more than series of injections over the months since we began this study. sfemg studies have revealed no significant change in the fiber density, mean mcd, or percent of fiber pairs with normal jitter in either muscle. in conclusion, our studies support the hypothesis that the presence of remote effects of botulinum toxin may be related, in part, to the amount of toxin used. the c bl/ / a mouse exhibits the remarkable characteristic of prolonged survival of axons separated from their cell bodies (slow wallerian degeneration). previous work has demonstrated that the axon itself is responsible for the phenotype of prolonged survival. we investigated whether the lack of rapid axonal degeneration after axotomy in this substrain is due to an inability to break down cytoskeletal components, a process that is normally accomplished by activation of intrinsic calcium proteases. segments of desheathed sciatic nerves from normal and ola mice were incubated for hours under conditions that disrupt the axolemma (freeze/ thaw or in % triton x-loo), allowing external calcium free access to axoplasm. nerves were analyzed by western blot for neurofilament (nf) proteins and by electron microscopy. in high-calcium media ( mm caci,), nf immunoreactivity was lost and axoplasm was reduced to watery debris in both substrains, whereas in egta-buffered media, axoplasm was preserved. these results demonstrate that calcium-activated proteases are present and can be activated in ola nerves. the defect in these mice that allows for prolonged survival of transected axons is likely in the mechanism for calcium entry into the distal stump. the mechanism by which the analogue of adrenocorticotrophic hormone, acta - , prevents cisplatinum (cp) neurotoxicity is unknown. murine n e. neuroblastoma cells and neural crest-derived, squirrel fish erythrophore cells tuesday, have similar vesicular transport mechanisms to human neural cells. they were used to study the effects of cp and acth , on cellular transport. differentiated n e. cells were treated hour prior to observation with serum-free media (sfm, control); sfm/cp pg/ml; or sfm/cp pg/ml and ng/ml acth -,. organelle transport was studied ( neurites and - organelles per condition) using computer-enhanced video microscopy. mean fast anterograde ( . k . pmsec-' vs . * . pmsec-') and retrograde ( . . pmsec-' vs . +- . pmsec-') transport were decreased in cp-treated compared to control cells ( p < . ). in cp/acth ,-treated cells, mean anterograde ( . +_ . pmsec-') and retrograde ( . . pmsec-') velocities were greater than in cp cells ( p < . ). velocities in control and cp/acth , cells were not statistically different. erythrophore pigment granule transport was observed in a blinded study, using similar techniques. mean aggregation velocity was greater in control ( . k . msec-') and cp/acth , ( . f . msec-')-treated cells compared to cp ( . * . msec-') cells ( p < . ). incubation with cp for or hours affected velocities equally, but acute exposure was more easily reversed by control or acth,, containing media. there is striking inhibition by cp in cross-species models of organelle transport. this can be prevented by acth ,. erythrophores allow future study of individual transport components. neurotrophin to investigate signal transduction pathways involved in neurite growth, the cytoplasmic regions of p sngfr, the common neurotrophin receptor monomer, were searched for a motif analogous to the predicted secondary structure of the tetradecapeptide mastoparan. potential sequences were modeled using a semi-empirical molecular mechanical force field approach. the sequence rat ~ ~~~ - represents a highly conserved amphiphilic domain predicted to be involved in neurotrophin signal transduction via g-protein mechanisms. to test this prediction, peptides containing sequences homologous to p ngfr - were examined for effects on trophic factor-induced survival/differentiation responses of rat pc pheochromocytoma cells, chick embryo drg neurons, and chick embryo ciliary neurons. a peptide identical to ~ ~~~ - accelerated the neurite growth response to nerve growth factor (ngf) of pc cells and drg neurons in a time frame that paralleled uptake into cells, but mastoparan did not influence ngf-mediated neurite growth. millimolar mg+ + and benzalkonium chloride, known to block the actions of mastoparan, blocked the effect of the peptide on ngf-mediated neurite growth by pc cells. peptides mutated to alter cationic amino acid relationships or amphiphilicity were less effective than the peptide in accelerating ngf-mediated neurite growth. these observations complement and extend evidence suggesting a pivotal role of ~ ~~~ in ngf-mediated signal transduction. these studies complement and extend evidence suggesting a pivotal role of p sngfr in neurotrophin signal transduction and evidence that activation of intracellular signalling processes involving specific g-protein mechanisms are involved in neurotrophin-mediated neurite growth. crushing the hypoglossal nerve causes hypoglossal motor neurons to decrease expression of choline acetyltransferase (chat) and begin expressing p ngfr, the low-affinity ngf receptor. these changes are evident within days after the injury and continue for several weeks. inhibition of axonal transport by vincristine applied to uninjured nerves causes loss of chat expression without induction of ~ ~~" . we sought to determine if topical vincristine would alter p sngf' expression after nerve injury. hypoglossal nerves were surgically exposed unilaterally in anesthetized rats and crushed. one week later, rats were reanesthetized and the same nerves were re-exposed. vincristine or saline was applied at the crush sites by soaking a strip of cotronoid wrapped around the nerves. one week later, rats were anesthetized and perfused with aldehydes. frozen sections from the brainstems were stained by indirect immunoperoxidase to demonstrate chat and ~ ~~~' . saline-treated controls showed decreased chat and abundant ~ ~~" in hypoglossal motor neurons ipsilateral to the crush injury. vincristine-treated animals showed no chat and no p tngf'. we interpret these results as indicating that a signal originating from the injury site maintains ~ ~~" expression after nerve injury. catecholamines have been reported to be toxic to embryonic-derived rat neurons and glia via the formation of reactive oxygen species (rosenberg, ) . we tried to determine whether oligodendrocytes (ol) from adult -month-old rat brain are similarly susceptible. toxicity to ol was examined using light microscopy and galactocerebroside immunohistochemistry where the relative number of surviving ol and their extent of process formation were graded. five days of exposure to norepinephrine (ne) and epinephrine (epi) at and fm produced significant toxicity ( p < . , analysis of variance [anova)) to adult rat ol; this toxicity was evident by hours of exposure. treatment with catalase ( p,g/ml), a free-radical scavenger enzyme, completely prevented the toxicity of catecholamines. to ascertain whether astrocytes, which have free-radical scavenging capacity, could prevent the catecholamine-induced injury to ol, rat ol were seeded on neonatal rat astrocytes. under such conditions, the toxicity of n e and epi was reduced significantly ( p < . , anova). these findings suggest that impairment of this protective function of astrocytes may render ol and its myelin membrane susceptible to free-radical-mediated damage. lyme neuroborreliosis is an increasingly prevalent disorder, but the diagnosis generally has been indirect. thus, the pres-ence of other manifestations of the infection, consistent findings on neurological exam or lumbar puncture, or presence of csf antibody have been used rather than direct isolation or identification of the organism from the csf. we have previously developed polymerase chain reaction with hybridization (pcr/h) to identify borrelia burgdorferi in the blood and organs of infected mice, and found that the assay was equivalent or, in some cases, preferable to culture (ann neurol ; : ) . the assay used for primers oligos derived from a sequence of genomic b. burgdovfri d n a expressed on a plasmid by rosa and schwan. a two-stage nested pcr was performed on csf samples in which the d n a was isolated in a variety of ways. pcr products were subsequently hybridized with a digoxigenin-labeled internal probe by slotblot hybridization. the sensitivity of the assay was excellent, being <o. fg of purified b. burgdoorfevi dnaiml of csf, and to spirochetes per ml of csf when "spiked" csf was tested. the assay then was applied to a battery of csfs stored over the past years in patients with definite, probable, and possible lyme neuroborreliosis. the lack of pcr/h positivity in some patients with definite and probable disease signifies that the spirochete does not reside in the lumbar csf in all patients with lyme neuroborreliosis, and may be concentrated in the meninges or parenchyma. progenitor cell line on transplantation into the adult murine brain t . kitagwcbi, d.-h. chui, and t . tabira, tokyo, japan several multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into primary culture cells of the septum of embryonic murine brains (the retroviral vector, kindly provided by d r c. l. cepko, boston, ma). two of those cell lines, when transplanted back into to -week-old mice by stereotaxic operation, integrated into the septum in a nontumorigenic manner. the implanted cells, which had been labeled with pkh dye, could be identified in animals up to at least weeks after transplantation. immunocytochemical analysis demonstrated that the transplanted cells were seen assuming a round morphology and no processes. they did not stain with any glial or neuronal markers except for a b and hnk-i, in the same way as in vitro. interestingly, after weeks of implantation, the cells were located discretely in the transplanted site and displayed a small and round morphology with fine processes. most of the transplanted cells showed immunoreactivity with monoclonal antibodies directed against neuronal markers such as neurofilament and map . these data indicate that the immortalized cell lines might be useful in characterizing factors that regulate cell type differentiation in the mammalian nervous system. (supported by a grant from the science and technology agency of japan.) related to serum response factor in human brain and muscle dana leifer, dimitri krainc, racbael neve, roger bveitbavt, and stuart a. lipton, boston, m a we have identified cdna clones for a novel transcription factor that is expressed at high levels in human fetal brain and skeletal muscle, but not in a variety of other tissues, as demonstrated by northern blotting. sequence analysis indicates that the clones appear to have alternatively spliced forms and have extensive homology with human serum re-sponse factor (srf) and a family of related transcription factors that has representatives throughout eukaryotic evolution in species from yeast to man. srf is thought to have a role in regulation of immediate early genes and of muscle-specific genes. in gel mobility-shift assays, the srf-like proteins coded for by our clones bind specifically to the myocytespecific enhancer binding factor- (mef- ) regulatory element, a dna sequence that has so far been found to be functionally important in the promoter and enhancer regions of a variety of muscle-specific genes. moreover, our srf-like proteins specifically activate transcription of reporter genes containing the mef- element. given the abundant expression of our clones in human brain as well as in muscle, our results suggest that these proteins may have a significant role in development not only of muscle but also of brain. a host of quantitative eeg studies, most of which employed variations of the fast-fourier transform, have been made on isolated clinical entities such as the epilepsies, metabolic syndromes, and sleep with varying degrees of satisfactory correlations. using a time domain wave-by-wave computer program that simulated the logical approach of the clinical electroencephalographer, we have studied more than unselected routine neurological clinic and ward patients. the presenting problems ranged from headache to major cerebral involvement. employing the dichotomy of normal and abnormal, the computer-read eegs agreed with the routine eeg, the clinical manifestations (including imaging techniques), and neuropathological findings in % of all cases. this high level of correlation, which approached that achieved by welltrained eegers reading the same records, was obtained by solely utilizing the background eeg activity. such results became possible only when the computer program was so organized that the "raw" computed and original oscillograph records were made to match closely. the diagnosis, with topographically diagramed localization, was printed automatically as the final step of the program. our data strongly suggest that routine computer reading of the human eeg is feasible, reliable, and potentially useful. protein accumulation in damaged neurites and microglial cells abnormal accumulation or abnormal processing of amyloid precursor protein (app), or both, may be critical to the development of p-amyloid protein (bap) deposits. however, alzheimer's disease (ad) has another pathological hallmark, which is the appearance of neurofibrillary tangles. the question is, therefore, to clarify the possible relationship between altered app metabolism and neurofibrillary degeneration. one classic method for inducing neurofibrillary tangles is the administration of aluminum salts. although these tangles are morphologically different than those seen in ad brain, this phenomenon has given rise to the hypothesis that aluminum might be an environmental factor in the causation of ad. this theory is controversial, but the animal aluminum model remains one of the few ways that long-lasting neuroskeletal changes can be induced. to explore the effects of aluminum salts on app metabolism, we examined app immunodistribution in rat brain injected intraventricularly or intrastriatally with aici,. we found a long-lasting accumulation of app in affected neurites, as well as in activated microglia/macrophages. abnormal neurites also showed argentophilic changes, neurofilament accumulation, and alz- immunoreactivity. the results support the hypothesis that interruption of axoplasmic flow by aluminum salts, as by other means, can lead to both app accumulation and neuroskeletal alterations. p . nondemyelinating conduction slowing of myelinated fibers due to inactivation of n a channel takanori yokota, yukinobu saito, and tadashi miyatake, tokyo, japan in patients with acute or chronic inflammatory demyelinating polyradiculoneuropathy, the marked improvement of motor-nerve conduction velocity without change of amplitude or configuration of the compound muscle action potentials (cmaps) was observed in days. to investigate the mechanism of the rapid improvement of nerve conduction, the influence of na channel blockade on nerve conduction was studied. in healthy volunteers, the recovery of the cmap and sensory nerve action potential (snap) of median nerve stimulation were recorded after the intravenous infusion of mg of lidocaine. after the loading, cmap and snap were reduced rapidly in amplitude and were slowed in latency. after the recovery of amplitude of cmap and snap, the nerve conduction velocities were improved gradually in hours with the amplitudes and configurations of cmap and snap unchanged. this indicated that myelinated nerve conduction velocity could be slowed by the inactivation of na channels without demyelination or conduction block. the prolongation of rising time in depolarization of axonal membrane potential should be one of the mechanisms of this conduction slowing due to inactivation of na channels. paraneoplastic cerebellar degeneration (pcd) in gynecological and breast cancers frequently is accompanied by an autoantibody response (type i or "anti-yo") reacting with and -kd cytoplasmic antigens of cerebellar purkinje cells. sakai et al, using a cerebellar library (stratagene), recently have isolated a cdna clone expressing a -kd protein recognized by igg from a patient with pcd and uterine carcinoma (sakai et al, ann neurol ; : ) . this clone is believed to share some homology with the message encoding the -kd protein recognized by type i sera. because pcd was isolated using serum from a single patient, however, it is not known whether the expressed protein of pcd is recognized uniformly by all patients with type i antibody response. e. coli strain xl -blue transformed with pwr - containing the snabiihindiii fragment of pcd , pwrsbo-pcdl?sn, was induced using isopropylthiogalactoside. bacterial lysates were electrophoresed on % sds-page gels and analyzed by western immunoblot program and abstracts, american neurological association methods using sera and csf from patients with pcd associated with gynecological and breast malignancies who exhibited type i antibody response. the -kd expression product was labeled by sera and csf samples tested from antibody-positive patients but was not labeled by sera or csfs from controls. our studies demonstrate that the -kd protein expressed by pcd contains epitopes that are consistently recognized by both systemically and intrathecally produced antibodies from patients with pcd accompanying gynecological and breast malignancies. tax is a novel antitumor drug that has demonstrated impressive clinical activity in breast, ovarian, and lung cancers. although sensory neuropath y has been described secondary to both taxol alone and taxol-cisplatin combination, detailed neurophysiological and pathological studies have not been described. nineteen patients with solid tumors were treated with combinations of taxol ( - mg/m ), cisplatin ( - mg/m ), and granulocyte colony-stimulating factor (g-csf) ( pglkglday). sequential neurological examinations, nerve conduction studies (ncs), and quantitative vibration testing (qst) were performed during and after treatment. eighteen of patients developed sensorimotor neuropathy and developed myopathy. thirteen were symptomatic with numbness or weakness and had abnormal results on neuromuscular examination. ncs showed absent or prolonged h-reflexes ( ), reduced peroneal motor amplitude ( ), reduced sural sensory amplitude ( ), and myopathic motor unit potentials in proximal muscles ( ). qst results were abnormal in of tested patients. muscle biopsy specimen in a patient with myopathy showed features of a toxic myopathy. the neuropathy was worse with higher cumulative dosages of taxol (> mg), with higher single doses of taxol(> mg/m ), or with a preexisting neuropathy. we conclude that sensorimotor neuropathy and myopathy are dose-limiting neurotoxicities of combined cisplatin and taxol use, now that neutropenia can be controlled with neuropathy and myopathy g-csf. central nervous system lymphoma casilda balmacedz and lisa deangelis, new york, ny ally periventricular and may seed the csf by direct growth through the ependyma. we reviewed the csf profile of non-acquired immunodeficiency syndrome (aids) patients (pts) with pcnsl. all pts had lumbar puncture (lp) and had multiple samples from an ommaya reservoir. definite lm involvement was identified with a positive csf cytology, lymphomatous lm infiltration on a surgical specimen, or mri with gadolinium showing lm tumor. probable lm lymphoma was diagnosed in pts with suspicious or atypical csf cytology. there were women and men with a median age of (range - yr). at diagnosis, mean white blood cell count was /mm (range - , ); mean lumbar csf protein was mgldl (range - , ); and mean ventricular csf protein was mgldl (range - ). glucose was always normal. nineteen of pts sampled had oligoclonal bands, and / had elevated pz microglobulin. at diagnosis ( %) had an abnormal csf cytology: positive, suspicious, and atypical. one pt had pathological infiltration of the lm and had lm tumor on spine mri for a total of ( %) pts with definite or probable lm lymphoma. in pts with abnormal cytology, the abnormality was found in ( %) lps and / ( %) of the ommaya and ventricular specimens. in pts the lumbar cytology was the only abnormal specimen despite multiple ventricular samples, and in only the ventricular csf was abnormal. thirty-six of ( %) pts developed recurrent tumor afrer treatment. forty-two percent ( ) of all patients with relapse had lm recurrence. lm recurrence was accompanied by brain recurrence in pts, systemic in , ocular in , both systemic and ocular in , and isolated in . at diagnosis / patients received treatment directed against the lm. of these, ( %) had meningeal recurrence whereas of the ( %) patients who did not receive this treatment had lm recurrence. lm involvement by pcnsl is frequent, may be missed on a single csf sample, and requires specific therapy at diagnosis. sixty-three solid cancer patients with a single brain metastasis were prospectively randomized for neurosurgery and radiotherapy combined (arm ) or radiotherapy alone (arm ). they were stratified for lung or nonlung cancer and for active versus stable or absent extracranial disease. world health organization performance status was . age, sex, performance status, and location of brain metastasis were divided evenly over both groups. one-month mortality was % in arm and % in arm . median survival of months after combination therapy was significantly better compared to months after irradiation alone ( p < . ). it made no difference whether they had lung or nonlung cancer. the largest difference between both treatment arms was observed in patients with stable or absent extracranial disease ( vs mo, p < . ). when systemic disease activity was present, median survival was months irrespective of treatment arm. functional independent survival was to months shorter than overall survival and was significantly better for patients with stable extracranial disease after combined therapy. multivariate analysis showed that age was also an independent prognostic factor. patients older than years had a hazard ratio for dying of . (p. ). we will detail the type and pattern of neurological complications in t-cell non-hodgkin's lymphoma (nhl), and review how they differ from those associated with b-cell nhl, and the lymphomas in general. this study is the first step in a process to characterize these tumors to determine if special staging or cns prophylaxis are indicated in any of the subtypes of t-cell lymphoma. we recently have encountered women with breast cancer and an unusual sensorimotor neuropathy. the neuropathy was the major clinical problem. in women the initial symptom was severe itching, generalized and first localized to the involved breast and then generalized. all developed distal extremity numbness and burning that very slowly progressed proximally, and in became generalized. four complained of painful muscle cramps in the extremities ( ) and jaw ( ). all had mild extremity weakness, distal ( ) and proximal ( ) . three women developed symptoms up to months prior to cancer diagnosis, shortly after diagnosis, and years after diagnosis. four women had disease confined to the breast and regional lymph nodes, and had metastatic disease in remission. although annoying, symptoms were generally not disabling. three women stabilized or had slight improve-associated with breast cancer ment with cancer treatment, and continue to gradually progress while in cancer remission; required a cane to ambulate after years due to sensory ataxia. one who developed cancer relapse had concurrent neurological relapse. one woman treated with high-dose immunoglobulin did not improve. none had significant weight loss. laboratory abnormalities included elevated erythrocyte sedimentation rate ( - ) in , antinuclear antibody : in , csf with lymphocytic pleocytosis ( - white blood cellslmm) in / and elevated protein ( - mg/dl) in available. emgi ncv showed mild sensory-to-motor polyneuropathy in available. none had detectable antibodies against peripheral nerve or dorsal root ganglia. the etiology of sensorimotor neuropathy in these patients is unknown, but it may represent a distinct paraneoplastic syndrome that can herald the onset of, and parallel the course of breast cancer. our objective was to determine whether ceramide induces differentiation of anaplastic glioma cells. sphingomyelin hydrolysis resulting in ceramide production has been linked to differentiation of leukemia cells. t rat anaplastic glioma cells, seeded at x lo cells per well, were grown in serum-a glioma cell line program and abstracts, american neurological association free media. on day , cells were photographed, scored for processes longer than one cell body, and counted. c ceramide changed plump cells to flattened cells with many long processes: ceramide treatment increased the percentage of cells with processes from % (sd) to * % (n = , p < . ). control cells grew to . x lo cells/well . x lo cells; ceramide-treated cells grew to . x lo ? . x los (n = , p < . ). although one ceramide analog reproduced the c ceramide-associated changes, the optical isomer of this analog did not, demonstrating stereospecificity. cerarnide did not decrease cell viability by trypan blue. ceramide inhibits proliferation and induces process formation in a glioma cell line, causing it to assume a more differentiated phenotype. cerarnide or its analogs represent possible future therapeutic agents that would inhibit the growth and affect differentiation of anaplastic gliomas. [bashir, mod pathol ; ( ) : - )). this is similar to the pattern seen in ebv-infected human b cells and unlike the uniform latent infection seen in burkitt's lymphoma. we tested the hypothesis that long-term passaging of ebv-immortalized human b cells in immunodeficient mice leads to emergence of a uniform nonlytic pattern of ebv infection associated with appearance of the malignant profile. ebv-infected normal human b cells were serially passaged intracerebrally in severe combined immunodeficient cb mice ( x ' cells per mouse, mice per passage for a total of passages). frozen mouse brain sections from each passage were stained with vca antibody (ebv lytic cycle) and hybridized with biotinylated bamh -w sequence of ebv. all injected animals developed tumors as previously described (bashir, lab invest ; ( ): - ) . tumor cells continued to express vca and showed latent and lytic hybridization patterns with bamh,-w after passages despite exhibiting monoclonality (surface immunoglobulins) and random chromosomal changes. lytic infection of immortalized b cells with ebv is stable, resembling brain lymphomas in aids, and unlike the latent infection seen in burkitt's lymphoma. a previously healthy -year-old man developed diplopia and incapacitating, diffuse weakness over a period of weeks. examination showed a "one-and-a-half'' syndrome of horizontal gaze paresis, patchy severe weakness with atrophy and fasciculations, absent tendon reflexes in the legs and right biceps, and decreased vibration sense in the feet. csf contained a mild pleocytosis, elevated protein, and oligoclonal igg bands. electrophysiological testing indicated a generalized sensorimotor axonal neuropathy with diffuse denervation. small-cell lung carcinoma was diagnosed by bronchoscopy. prednisone produced mild subjective improvement. chemotherapy was begun but the patient developed fatal septicemia. serum was negative for anti-hu or anti-gm with paraneoplastic encephalomyeloneuritis antibodies. serum and csf contained high titers of igg antibodies reacting specifically with a protein antigen of approximately kd in immunoblots of human cerebral cortical neuronal nuclei or of human purkinje cells. this pattern of autoantibody reactivity was not present in sera from any of other patients with small-cell lung carcinoma, of whom had paraneoplastic encephalomyeloneuritis and anti-hu antibodies, nor was it present in many patients with other neurological disorders. the patient's serum has been used to probe a human cerebellum expression library and to isolate a cdna clone that is being characterized. acute encephalopathy is the problem in % of neurology consultations reported at mskcc. we studied patients ( prospectively and retrospectively) to determine clinical findings, causes, and outcome. fifty-five were women and were men, and the average age was years. all patients had cancer: lung ( %), gastrointestinal tract ( %), breast ( %), and others ( %), forty-two patients ( %) were delirious on admission and delirium developed an average of days later in %. encephalopathy occurred postoperatively in %. symptoms included confusion ( %), lethargy ( %), agitation ( %), hallucinations ( %), and seizures ( %). signs included deficits in attention ( %), memory ( %), language ( %), lateralizing signs ( %), and asterixis ( %). the average mini-mental status test (mms) score was ( = normal). a single cause for delirium was found in only % of patients with an average of etiologies per patient. metabolic abnormalities were found in % of patients, and were a primary cause in %; disseminated intravascular coagulation contributed to delirium in %. cns metastases were found in % and were a major cause of delirium in all. fifty percent of the patients had fever/ systemic infection, but sepsis was present in only %; only patient had cns infection. medication contributed to delirium in % but was a primary cause in only %. the -day mortality rate was % and delirium improved in % (average mms = ). patients with cancer have multiple, potentially treatable causes of delirium. delirium is associated with a high death rate, though patients generally improve. radiation therapy for brain tumors d. w. dodick, b. mokri, k. k. unni, g. m. miller, and e. g. shaw, rochester, m n osteosarcoma in a previously normal bone is a rare but recognized remote effect of radiation therapy. any bone in the field of radiation can be affected. involvement of cranial bones is exceedingly rare. we could identify only patients ( men and women) with postirradiation osteosarcoma of the calvarium seen at the mayo clinic over a -year period, from to . all had received radiation for brain tumor, osteosarcoma had appeared in the field of radiation in all, the interval from radiation therapy to the appearance of sarcoma ranged from to years, and diagnosis of sarcoma was confirmed histologically in all cases. the patients' age at the diagnosis of the brain tumor ranged from to years. the nature of the brain tumor was unverified in cases, was a low-grade ependymoma in the third case, and a pilocystic astrocytoma in the fourth case. one patient is still alive months after the diagnosis of the sarcoma. she received chemotherapy and subsequently underwent resection of the osteosarcoma. one patient died postoperatively after partial resection of the sarcoma. the other patients died months and months after the diagnosis of the osteosarcoma despite additional radiation therapy in the former and aggressive chemotherapy in the latter. element-la fusion gene as a potential marker of neural tumor differentiation lawrence recht, chiffon wu, and louis j. degennam, worcester, ma inducing cancers to differentiate into more benign differentiated tumors represents a novel oncological strategy. to establish a model that would permit assessment of this phenomenon at a molecular level, we created and have partially characterized a murine neuroblastoma line that has been stably transfected with a synthetic fusion gene containing the promoter element of the rodent synapsin i gene (synapsin i regulatory element isre)). in vivo, this promoter directs the neuron-specific expression of the synapsin i gene in normal adults. the gene also is expressed in varying amounts in neuronal tumors including neuroblastoma. in the synthetic fusion gene, the sre has been linked to the lacz gene that encodes bacterial p-galactosidase. a simple histochemical assay for p-galactosidase therefore provides a specific marker of the expression of the fusion gene. our preliminary experiments as of this writing have shown that it is possible to detect p-galactosidase activity in the transfected neuroblastoma cells both in vitro and in transplanted tumors. it appears possible therefore that this transfected neuroblastoma cell line can provide a useful model system with which to assess the effects of differentiation therapies. larger lesions (> cm ), and larger midline shifts (> mm). twenty-eight of ( %) patients with prior has had bt has compared to of ( %) patients without prior has. in patients, their bt h a was similar to their previous ha but was more frequent or severe. we conclude that has in bt patients are common but usually not severe. nausea, vomiting, an abnormal neurological examination, or a change in prior headaches warrant further investigation. cairncross and macdonald showed that procarbazine, lomustine, and vincristine (pcv) are effective for recurrent anaplastic oligodendrogliomas (ao) (ann neurol ; : - ) . pcv now has a major role in management of all forms of oligodendrogliomas ( ), but the biological basis for this response is unknown. to evaluate one subset of possibilities, we studied patients ( ao, ) with a ct method that permits measurement of blood-to-tissue transport (kj, tumor-to-blood transport (k ), and vascular volume (v,) (ann neurol ; : - ) . pcv was used to treat of the patients. k, (~ grr-' min-') values were highly variable for whole tumor, ranging from . to . (mean . k . ) with no difference between a and . k and v, were also highly variable. k, of tumor-free brain was . to . (mean . ? . ). in comparison to malignant astrocytomas, which have a mean k, in the range of . with some as high as . , a and appear to be much less permeable. this suggests that the efficacy of pcv may be due to factors other than capillary transport, such as tumor-cell sensitivity. frameless stereotactic localizer gene h. barnett, donald w. komos, and charles p . steiner, cleveland, oh extent of tumor resection has been shown to correlate with prognosis in malignant gliomas. although frame-based stereotactic techniques can provide information regarding tumor margin, they are often unwieldy and require expensive and elaborate computing systems. a frameless stereotactic neurosurgical localizing system was designed that overcomes these liabilities. this armless, frameless, stereotactic pointing device provides real-time three-dimensional localization information during operation. in addition to assisting in placement of a trephine craniotomy, it allows volumetric resection of the tumor with virtually complete excision of even large irregularly shaped tumors. mean error on localizing a point in space using this system has proven to be less than mm. a technical description of the system as well as surgical results are presented. to investigate the effect of age on response rate to chemotherapy and time to progression (ttp) and death ('itd) in patients with recurrent astrocytomas and malignant astrocytomas, we reviewed case records and scans of patients who received chemotherapy at the university of michigan with bischloroethylnitrosourea or procarbazine. three age groups were studied: ( ) < yr (n = ); ( ) - yr (n = ); ( ) > yr (n = ). tumors were grouped as grade r+ (recurrent grade plus grade , n = ) or grade (n = ). serial computed tomographic or magnetic resonance scans were analyzed in a blinded fashion and graded as progressive disease (pd), stable disease (sd), or partial response (pr, > % decrease in size). the pr rates for the age groups were %/ %/ % for grade r+ tumors ( p = . ) and %/ %/ % for grade tumors ( p = . ). median 'itp was weeks for grade r + and / weeks for grade . median ltd was / / weeks for grade r+ and / / weeks for grade . we conclude that age is an important prognostic factor with respect to likelihood of response to chemotherapy, duration of response, and survival irrespective of grade. cheryl p. harris and kurt a. jaeckle, salt lake city, ut intravascular malignant lymphomatosis (iml), a b-cell lymphoma confined to small venules and capillaries, often presents with neurological symptoms. this disease is uniformly fatal ( -month mean survival); no successful treatment has been identified. we observed marked reproducible neurological improvement after plasmapheresis in a -year-old woman with iml. presenting with a cauda equina syndrome, she progressed over year with neurological, hepatic, and hematological disease. persistent laboratory abnormalities included a high sedimentation rate ( mm/hr), coagulopathy, hemolytic anemia, and elevated liver enzymes. extensive evaluations for infectious, autoimmune, and neoplastic processes, including bone marrow examination, were inconclusive. because of neurological progression, empiric therapy with high-dose steroids followed by cyclophosphamide was initiated without response. plasmapheresis ( ml/kg in exchanges) effected resolution of encephalopathy and normalization of the coagulopathy and sedimentation rate. neurological progression recurred within weeks of pheresis; repetitive courses reproduced neurological response. finally, progressive dementia ensued, and a decision was made to cease pheresis; the patient died days later, months after presentation. autopsy disclosed diffuse intravascular cd- positive malignant lymphoma cells in small vessels of all organs. although the mechanism is unknown, the serendipitous discovery of response to plasmapheresis in this patient warrants further consideration. morphine is an effective analgesic in the rat after injection into a number of discrete brainstem regions, including the periaqueductal gray (pag), the locus coeruleus (lc), and the nucleus raphe magnus (nrm). early work with morphine gray and the nucleus raphe magnus established the existence of synergy between the brainstem and the spinal cord in rats. more recently, studies from our laboratory revealed synergy between two brainstem structures, the pag and the lc. in the current study, we explored the analgesic interactions between the pag and the nrm using indwelling cannulae. first, we established morphine dose-response curves and calculated the ed,, independently in the pag ( . pg) and the nrm ( . pg). we then simultaneously injected various morphine doses into both regions. injecting morphine at pg into either the pag or the nrm did not elevate tailflick latencies above baseline values. however, administered into both regions simultaneously, the -pg doses produced an % maximal response, corresponding to more than a threefold increase of baseline latencies. a fixed morphine dose of pg in the pag shifted the morphine dose-response curve fivefold in the nrm (ed,, . pg), whereas a fixed nrm dose of fg shifted morphine's dose-response in the pag approximately twofold. together, these results clearly show synergistic interactions for morphine between the pag and the nrm. the presence of synergistic interactions between brainstem nuclei as well as between the brainstem and the spinal cord underscores the complexity of opioid analgesic systems. p . the glutamate uptake inhibitor ~-trans- , -pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain john d. e. barks and faye s. siloerstein, ann arbor, m i important evidence of the neurotoxicity of endogenous glutamate (glu) in mammalian brain was provided by the observation that dl-threo- -hydroxyaspartate, a high-affinity glutamate uptake (hagu) inhibitor, was neurotoxic in adult rodent striatum (j neurochem ; : ) ; however, the absence of neurotoxicity in neonatal brain was interpreted as evidence that immaturity of glutamatergic innervation limited the potential role of endogenous glu as a neurotoxin in the immature brain. yet, considerable data provide indirect support for the hypothesis that glu can be neurotoxic at this stage. to resolve this issue, we assessed the neurotoxicity of a novel, selective hagu inhibitor, ~-trans- , -pyrrolidine dicarboxylate (l-pdc) (j med chem ; : ), in postnatal day (pnd) rats (n = ). l-pdc (ph . ) was stereotaxically injected into right anterior striatum (str) ( nrnol, n = ) or through dorsal hippocampus into posterior str ( nmol, n = ; nmol, n = ). animals were killed days later, and neuropathology was assessed in cresyl violet-stained sections. after anterior injections, focal neuronal necrosis was evident in dorsal str; high-dose posterior injections caused prominent hippocampal lesions with pyramidal layer thinning and focal necrosis in dorsal thalamus, while nmol produced small foci of pyramidal cell loss. in both groups, focal cortical necrosis and callosal cysts were apparent adjacent to the injection track. l-pdc-induced brain injury provides direct support for the hypothesis that endogenous glu may be neurotoxic in the developing brain. (sax et al, ann neurol ; : a) . the signs and symptoms of of these patients lessened for to months. furthermore, a patient with a severe oral-lingual biting dyskinesia improved when taking doses up to milligrams per day for months. we noted no-significant adverse reactions, although patients had mild but labile elevations in sgop and sgpt. one patient receiving opioid analgesics for pain inadvertently failed to discontinue the naltrexone but noted no reduction in the pain-alleviating effects of the analgesic. although hyperkinesia, especially of midline functions, as well as quality of life improved for the hd patients, their cognitive deficits remained unaffected. these observations suggest that chronic naltrexone is a safe and effective agent to treat chorea, dysphagia, and oral dyskinesia in hd for periods longer than a year. furthermore, they indicate that naltrexone can be effective in ameliorating oral-lingual biting tardive dyskinesia. these findings support our previous hypothesis that endogenous opioids play a role in rhe modulation of the dopamine system in hyperkinetic stereotypic movement disorders. a. jon stoessl, elizabeth szczutkmuski, and hanna fydvszak, london, ontario, canada we previously have demonstrated (psychopharmacology ; : - ) that intraperitoneally (ip) administered cholecystokinin (cck)- s suppresses vacuous chewing mouth movements (vcms, a putative mode of tardive dyskinesia) in rats exposed to chronic neuroleptics. as cck is not thought to cross the blood-brain barrier in significant amounts, its site of action in this paradigm is unclear. other behavioral and neurochemical effects of ip cck are blocked by vagotomy. male sprague-dawley rats were administered fluphenazine decanoate (flu; mg/kg im) or its vehicle every weeks for approximately weeks. cck ( , , or ng intracerebroventricularly) had no effect on neuroleptic-induced vcms. another group of neuroleptic-treated rats was subjected to bilateral subdiaphragmatic vagotomy or a sham procedure. cck- s ( , , or pg/kg intraperitoneally) suppressed neuroleptic-induced vcms in shamoperated animals, which confirmed our previous results. in vagotomited animals, chronic flu failed to induce vcms and cck was without effect in vehicle-or neuroleptictreated animals. these data suggest that the effects of cck on flu-induced vcms may be mediated peripherally, and that vagal pathways may be important for generating this response. (supported by the ontario mental health foundation and the ontario ministry of health.) p . excitotoxic amino acids are not involved in dopaminergic neurotoxicity of m p t p eldad mehmed, jutta rosenthal, and avinoam reches, petah tiqva, tel aviv, and jerusalem, israel the dopaminergic (da) neurotoxicity of -methyl- phenyl- , , , -tetrahydropyridine (mptp) is mediated via its oxidation in cns to mpp + , which enters da neurons and poisons mitochondrial complex i. da neuronal damage induced by direct nigral mpp+ injection is prevented by pretreatment with n-methyl-d-aspartate receptor antagonists, which suggests that excitatory amino acids are involved in mptp toxicity. since local mpp + application may produce nonselective nigral damage, we examined whether excitotoxins have a role in toxicity of systemically administered mptp. c black mice were injected intraperitoneally, once, with mptp.hci( mg/kg) and decapitated days later. groups of animals underwent the following pretreatments: (i) decortication week prior to mftp; ( ) intracerebroventricular injections of the excitatory amino acid receptor antagonists -amino-phosphonoheptanoate and d-glutamyl-glycine; and ( ) intraperitoneal injections of the calcium channel antagonists nimodipine, diltiazem, and flunarizine minutes prior to mptp. mptp produced marked striatal da depletions. decortication, destroying glutamatergic corticostriatal projections, intracerebral amino acid receptor antagonists, and systemic calcium channel antagonists did not protect mice against mptp toxicity; mptp-induced striatal da decreases were similar to those given the neurotoxin alone. this study suggests that excitotoxins are not involved in the mechanism of mptp toxicity. scopolamine-induced cognitive deficits k . j . meador, m . e. allen, p. franke, e. e. moore, and d. w. loring, augusta, ga a unique neurophysiological role for thiamine in cholinergic systems has been suggested. total thiamine content in cholinergic nerve terminals is comparable to that of acetylcholine, and the phosphorylation state of thiamine changes with release of acetylcholine. thiamine binds to nicotinic receptors and may exhibit anticholinesterase activity. based on these observations, we investigated the effects of pharmacological doses of thiamine on the cognitive deficits induced by the anticholinergic scopolamine in healthy young adults using a randomized, double-blind, placebo-conrrolled, double crossover design. cognitive tests included the p eventrelated potential and free recall memory for a verbal paragraph. conditions included baseline (bl), thiamine gm by mouth and scopolamine . mg/kg intramuscularly (b + scop), and lactose by mouth and scopolamine (plac + scop). testing was performed hours post thiamine or placebo, and . hours post scopolamine. thiamine significantly reduced the adverse effects of scopolamine on p latency (f [i, = . , p . ) and percent recall memory ( f el, ) = . , p . ). means (+sd) and p latency (ms) were bl = ( ), b + scop = ( ), and plac neurol ; : - ) . we previously reported that cff improved in of ms patients ( %) given el- orally in divided daily doses ranging from . to . mg (stefoski et al, neurology ; : - . subsequent analysis revealed that in of the patients cff improvements and reversals followed in a phase-locked trend the rising and falling serum concentrations of el- , including patients whose changes remained below the % increase needed to qualify for the improved category. these results resemble the phase-locked effects of temperature on neurological function in ms. the cff changes, because they so closely reflect variations in serum concentration, suggest that el- tissue levels closely follow those in serum and that el- rapidly crosses the blood-brain barrier. efficacy of el- in ms is also predicted to have a close relationship to serum levels. p . development of a n internal standard detectable by proton and phosphorus- nmr and hplc w. e. klunk, k. panchalingam, r. j . mcclure, and j . w. pettegrnu, pittsburgh, pa comparison of quantitative results from different analytical techniques can prove difficult due to the peculiarities of the particular techniques and the lack of a common standard applicable to all of the techniques. recently, both in vivo and in vitro nuclear magnetic resonance (nmr) have been applied to the quantification of a large variety of metabolites. although nmr can be applied to the study of living tissue, the question arises of how this technique compares with more traditional techniques such as high-pressure liquid chromatography (hplc). although this question can be addressed partly by studying perchloric acid (pca) extracts, it is difficult to directly compare this in vitro nmr data with results from hplc. to address this question, we have developed an internal standard that can be quantified directly by in vitro phosphorus- nmr, proton nmr, and by -fluorenylmethyl chloroformate (fmoc) derivatization followed by separation by hplc. a variety of aminophosphonic acids were studied by 'p nmr, 'h nmr, and hplc. promising compounds were added to pca extracts of human brain. the optimal compound was found to be -aminopropylphosphonic acid (app, ho p-ch -ch,-ch -nh ). app is easily detectable by all techniques, falls well outside of the region of interest in phosphorus- nmr, and is well resolved by proton nmr at mhz. it is easily separable from the phosphomonoesters and phosphodiesters observable by hplc and from the amino acids that occur in brain in significant concentrations. app appears to be a useful internal standard in the study of phosphorus and amino acid metabolites by in vitro p nmr, 'h nmr, and hplc. theories of sentence comprehension hypothesize at least a grammatical component that establishes the relationship among words in a sentence, and a semantic component that determines the meanings of these words. we used positron emission tomography (pet) to quantify regional cerebral blood flow (rcbf) in neurologically intact subjects during their detection of a letter target, a grammatical target, or a semantic target in the same written sentences. a mixedmodel analysis of variance (anova) revealed significant main effects for region (f e , ) = . ; p < . ), condition (f , = . ;~ < . ), and a significant region times condition interaction ( f {go, = . ; p < . ), but there were no differences between individual subjects. subsequent anovas revealed increased rcbf in a unique set of brain regions during the subjects' response to a grammatical probe when compared to their response to a letter probe of the same sentences. a unique distribution of rcbf also distinguished response to the semantic probe from response to the grammatical probe and the letter probe. other brain regions apparently contributed to performance for several activation conditions. these findings support the hypothesized dissociation of specific linguistic components based on their unique cerebral topographical representation, and that a distributed network of brain regions subserves sentence comprehension. cerebral music processing-a comparison study of musically trained and naive individuals louis s. russo, jr, jachonville, fl we performed topographical mapping of brain electrical activity in right-handed symphony musicians and righthanded, musically naive individuals during various musical tasks; namely, listening to solo piano music, silent singing of familiar music, and silent reading of unfamiliar music. fast-fourier transform ( f r ) of electrocortical activity was carried out during task performance and the eyes-open resting state. data were analyzed using a computer-assisted model. increases in regional beta activity of greater than standard deviations from the resting state were considered significant of activation. during audition, the musicians showed activation in the right posterior parietotemporal region; the naive showed no change from the resting state. during silent singing, the musicians showed bitemporal activation, r > l; the naive showed activation in the right mid-and posteriortemporal regions alone. during silent sight reading, the musicians showed a major activation in both temporal regions, l > > r, the naive showed only a marginal change in the posterior temporal-occipital regions. these data suggest that music processing is primarily a right cerebral function in untrained individuals and a bilateral function in musicians. musicians, in contrast to the naive, show progressively more left brain activation as task complexity increases. the present study analyzes language profiles in patients who presented with primary progressive aphasia (ppa) without global dementia for at least years. language and cognitive impairment were evaluated using the western aphasia battery (wab) and the mattis dementia rating scale (drs). expressive language disability with reduced speech fluency and anomia, but preserved language comprehension and nonverbal cognition, were typical features in early stages. spontaneous speech was significantly more impaired in ppa than in anomic aphasia after left-hemisphere stroke and in language impairment in probable alzheimer's disease (ad) ( p = . ). the profile of aphasia suggests that ppa tends to affect anterior parts of the language-dominant cortex first. neuroimaging generally showed mild to moderate brain atrophy. in patients atrophy involved especially the left frontal progressive aphasia cortex. follow-up examinations that were done in patients or several years after the first assessment revealed continuous, most often rapid deterioration of language impairment. two patients died and years after the onset of ppa. neuropathological examination showed ad in patient and pick's disease in the other patient. beverly clarke, adrian upton, markad kamath, and helene griff;., hamilton, ontario, canada eight patients implanted with a cyberonics neurocybernetic prosthesis model to stimulate the vagus nerve were assessed for changes in cognitive performance. the patients had complex partial seizures for more than years, with more than per month. patients were years * . sd old. cognitive evaluation included response time to a randomized light signal appearing on a switch box (test a); test b, in which the signal appeared bilaterally; and test c, in which a response to the signal was required while the patient simultaneously ignored a second signal. data were collected and analyzed using an apple i e computer and switch pad. all patients were taking therapeutic levels of anticonvulsant medications and dosages were constant. testing occurred times during a day preoperatively (day l), weeks postoperatively with the stimulator on (day ), and months after turn-on (day ). patients were randomized into high-and low-frequency stimulation groups (hfg and lfg). hfg parameters were hz, so msec pulse width (pw), and lfg hz, msec pw. examiners were blinded as to group. student's t-test analyses of mean differences between groups and individual measurements showed a significant difference between hfg and lfg for test c ( p < . ). lfg showed a significant improvement for tests a, b, c between day and , and for test c between day and . no group effect was seen between day and in the lfg. individual measurements showed improvement for test b ( p < . ) for the hfg between day and , test b ( p < . s) between day and , and tests a and b ( p < . ) and ( p < . ) day vs . the lfg group improved between days and and and . between day and day , the lfg showed improvement only for test b ( p < . ). chronic stimulation of the vagus nerve improves cognitive function in epileptic patients and this improvement is more marked with low-frequency stimulation. a. guidotti, and j. d. rothstein, bologna, itah, washington, dc, and baltimore, m d we reported idiopathic recurring stupor (irs) in a patient with stuporous episodes without known causes and reversed by flumazenil, a specific benzodiazepine (bz) antagonist. ictal plasmdcerebrospinal fluid (csf) showed increased bz-like activity (ann neurol, in press). recently, an endogenous bzreceptor ligand (endozepine [ez]) has been purified from mammalian brain with properties similar to diazepam. it acts like diazepam to potentiate gamma-aminobutyric acidmediated postsynaptic inhibition. we hypothesized that irs might be due to an excess of this substance. irs was diagnosed in patients, and years old, who had recurring stupor or coma episodes lasting hours to days. ictal brain ct/mri, kidney, liver, heart, blood glucose, ammonia, and osmolality were normal. eeg showed fast -hz background activity while the patients were unreactive to stimuli, reversed by flumazenil. ictal serum or csf revealed an enormous increase of the ez in both patients, with levels as high as nm, compared to to nm in control serum/csf. interictal csf or serum in irs contained ez levels similar to control csf and serum. irs may be due to excess ez. the cause for increased ez is unknown. parkinson's disease: evidence from word learning murray grossman, jenger mickanin, barbara schaefr, kris onishi, matthew b. stern, steven gollomp, and howard hurtig, philadelphia, pa several reports have suggested that patients with parkinson's disease (pd) have intellectual impairments in several domains such as memory, but few studies have explored difficulties in language processing. we investigated the ability of nondemented pd patients with mild motor impairments to learn about the grammatical and semantic information represented in a new verb. the new verb was presented to patients in a sentence-picture matching context, and we probed their recall of the verb minutes later. a sentence judgment task assessed grammatical knowledge by asking patients to judge the new verb, known verbs, and pseudowords used appropriately or incorrectly in a sentence. we found that % of pd patients were significantly impaired in their grammatical appreciation of the new verb (f [i, = . ;p < . ). this was not related to their motor disorder or neuropsychological performance. a picture classification task used pictures illustrating specific aspects of the new word's meaning to evaluate semantic knowledge. pd patients were as accurate as controls at deciding whether a picture illustrated the meaning of the new verb (f ( , = .lo;p > . ). only pd patient ( %) had difficulty sorting pictures. selective difficulty recalling only grammatical aspects of a new word suggests that the word learning impairment in pd cannot be entirely explained by poor memory. instead, in agreement with other recent findings, pd patients may be impaired in some aspect of grammatical processing in language. we discuss the hypothesis that defects in the frontocaudate axis in pd underlie this impairment. neuropsychological performance on both verbal and nonverbal tasks is reported to differ between healthy men and women. some of these cognitive differences are postulated to reflect differences in interhemispheric and intrahemispheric cerebral organization. our preliminary study indicated that women with alzheimer's disease (ad) performed worse than men on a composite neuropsychological battery, even after effects of potentially confounding variables were considered (buckwalter et al, j clin exp neuropsychol ; : ) . to explore further the nature of gender-associated differences in ad, we analyzed data from a verbal and a nonverbal task (the boston naming test and drawings from the spatial quantitative battery supplement to the boston diagnostic aphasia examination) for men and women who met nincds-adrda criteria for "probable" ad. prior to ana-grip strength kg [range . - kg]). the electrophysiological examination showed improvement with reversal of conduction block in patients and was unchanged in . an apparent response to the placebo was seen in patients. improvement after ivig therapy was maintained for variable durations ( - wk) and reoccurred with subsequent infusions. an equally effective response was documented after infusion of a single ivig dose of gm/kg. we conclude that ivig therapy is effective in some patients with cidp, even after long duration of illness. the best responses were observed in patients with recent relapse. a single high-dose treatment may be equally effective. the object of this study was to examine whether borrelia burgdorjeri antigens could be detected in csf in the absence of detectable antibodies to b. burgdorjeri (the etiological agent of lyme disease). osp a is a -kd antigen that is specific for . duvgdorferi osp a was probed using western (immuno) blot and specific mouse monoclonal antibodies. polyclonal lyme antibodies were detected in csf using standard micro enzyme-linked immunosorbent assay. seven patients had osp a in csf without detectable lyme antibodies. there were men and women aged to years (mean yr). disease duration ranged from weeks to years. neurological syndromes included confusion with acute flulike illness, optic neuritis, hemiparesis with inflammatory brain lesion, encephalitis, headache with erythema migrans, bilateral facial nerve palsies, and encephalomyeloradiculitis. three patients had csf abnormalities. in patients csf parameters were otherwise completely normal. possible explanations for undetectable csf lyme antibodies included early infection ( patients), prior antibiotics ( patients), and prior steroids plus antibiotics ( patient). in patient there was no obvious explanation. we conclude that osp a, a specific antigen of b. burgdorferi, may be present in csf without a detectable humoral response. the diagnosis of neurological infection with b. bwgdorjeri should not require a positive csf serology. mollaret's meningitis: demonstration by polymerase chain reaction a -year-old man was seen in september for his fourth episode of aseptic meningitis over a -year period. the episodes conformed to criteria for mollaret's meningitis as published by bruyn, straathof, and raymakers, and subsequently by others; they lasted about week, and were characterized by fever, headache, meningismus, lymphocytic pleocytosis, elevated protein in cerebrospinal fluid (csf), and spontaneous resolution without residua. extensive prior evaluations had failed to uncover a cause. the patient was otherwise well and neither he nor his wife had any history of sexually transmitted diseases. suspicion of herpes simplex virus (hsv) arose due to a single transient, raised skin rash several weeks earlier that failed to yield virus on culture. the patient was treated with acyclovir, which resulted in rapid resolution of symptoms. though culture and immunological studies of csf and blood again were unrevealing, polymerase chain reaction (pcr) studies of csf confirmed the presence of hsv type . we suspect that herpes simplex virus is a more common cause of recurrent aseptic meningitis than current culture and immunological techniques would suggest. pcr offers increased diagnostic sensitivity for neurotropic viruses and should be considered in patients with recurrent meningitis of cryptic etiology. differentiation by inorganic lead: a role for protein kinase c j. lutewa, j. p. bressler, r. r. indurti, l. belloni-olivi, and g. w . goldstein, baltimore, m d microvascular endothelial function in developing brain is altered by inorganic lead. this may result from changes in protein kinase c (pkc) modulation. we examined the effects of inorganic lead on an in vitro model of neural endothelial differentiation. astroglial-induced endothelial differentiation into capillary-like structures was inhibited by lead acetate with % maximal inhibition occurring at . pm lead. inhibition was independent of effects on cell viability or growth. we examined the effects of lead on cellular pkc pools under conditions that inhibited capillary-like structure formation. membranous pkc increased in c astroglial and neural endothelial cells after exposure to lead acetate. exposing c cells to p,m lead for hours increased membranous pkc by % as determined by immunoblotting. membranous pkc increased in response to as little as nm lead and saturated at pm. phorbol esters were used to determine if pkc modulation was mechanistically related to lead's inhibition of capillary-like structure formation. -myristate -acetate ( nm) inhibited endothelial differentiation by * %, whereas -alpha-phorbol , didecanoate was without effect. these findings demonstrate that inorganic lead may induce cerebral microvessel dysfunction by interfering with pkc modulation in microvascular endothelial or perivascular astroglial cells. w. f. brown, b. v . watson, j . garland, g . c. ebers, and n . desai, london, ontario, canada gait difficulties in multiple sclerosis (ms) are commonly accompanied by fatigue and dyspnea. possible explanations for the latter include weakness andlor dyssynergia of the respiratory muscles, including possible abnormalities in central pathways regulating respiration. this study examined central and peripheral motor conduction to the diaphragm in ms patients whose gait was notably labored and accompanied by breathlessness. peripheral conduction was assessed by measuring the latency and size of the surface-recorded diaphragmatic maximum m-potential responses to supramaximal stimulation of the phrenic nerve in the neck, and central motor conduction by comparable measurements in response to magnetoelectrical stimulation over the vertex. peripheral motor conduction was normal. the most striking abnormalities were in central motor conduction. cortical stimulus-evoked diaphragmatic responses were absent on both sides in patients, and unilaterally in l patient, whereas in others the latency of the cortical stimulus-evoked response was increased and the size clearly reduced and entirely normal in only patients. these studies show that central conduction program and abstracts, american neurological association to the diaphragm is commonly abnormal and may play a role in the fatigue and dyspnea experienced by ms patients. six men ( - yr) developed myelopathy that progressed slowly over several months and was characterized by asymmetrical, incomplete spinal cord syndrome manifested at the sensory level at the trunk, mild spastic paraparesis, and urinary incontinence. the spinal cord lesions at appropriate levels were recognized by mri as enhancing lesions in of the men. coxiella burnetii infection was confirmed in the blood of all patients by immunofluorescence microscopic assay (ifa) and transmission electron microscopy (tem). in patients, we detected c. burnetti by tem and ifa using csf of the patients inoculated onto fresh peripheral blood lymphocytes. four patients who were treated with appropriate antibiotics responded with either partial resolution of symptoms or arrest of further neurological progression. in patients the lesion was shown on mri to have decreased in size. in summary, we report cases of transverse myelopathy associated with c. barnetti infection. this is the first report, to our knowledge, of coxiella-related chronic myelopathy. we present a series of patients with different labyrinthine lesions diagnosed by mri. twelve patients with sensorineural hearing loss were studied by gadolinium-enhanced mri, including -mm contiguous t -weighted images through the labyrinth. ten patients had enhancement of the cochlea or vestibule, or both. all patients with cochlear enhancement had severe neural sensory hearing loss. all patients with vestibular enhancement had severe vestibular symptoms. the patients' final diagnosis included viral labyrinthitis ( patients), syphilitic labyrinthitis ( patients), bacterial labyrinthitis ( patient), and vestibular neuromas ( patients). one patient had an acoustic neuroma extending in the basal turn of the cochlea. the enhancement in patients with vestibular neuromas was brighter and there was slight mass effect in comparison with the patients with inflammatory labyrinthine lesions. one patient had hemorrhage within the vestibule from an adjacent temporal bone hemangioma. one patient with ct-proven cochlear otosclerosis had pericochlear areas of enhancement on gadolinium mri. mri can diagnose a variety of labyrinthine lesions that correlate very well with the patient's clinical symptoms. gadolinium should be used routinely in patients with suspected labyrinthine disease. the diagnosis of meniere's disease and endolymphatic hydrops remains a diagnosis of exclusion. few radiographic findings have been correlated with the clinical symptoms of this entity. we describe patients with symptoms of hearing loss or vertigo, or both, who demonstrated enhancement of the endolymphatic sac on gadolinium-enhanced mri. no enhancement was noted in a series of controls with no symptoms of hearing loss and vertigo. enhancement in the brain correlates with inflammatory or neoplastic conditions. we thus can speculate that enhancement of the endolymphatic sac reflects an inflammatory process in this location that may interfere with the normal resorption of indulin and secondary hydrops. in addition to excluding an acoustic neuroma and a labyrinthine schwannoma (which clinically may be confused with meniere's disease), contrast-enhanced mri may provide objective evidence in favor of labyrinthine hydrops. it was intermittent ( %) but progressive. the ha was mild to moderate in severity; it was the worst symptom in only ( %) and the first symptom in ( %) patients. has were worse in the morning in ( %) and interfered with sleep in ( %) patients. unlike true tension-type has, bt has were worse with bending over in ( %), with valsalva's maneuver in ( %), and nausea or vomiting were present in ( %) patients. an abnormal neurological exam was found in ( %) patients with has and ( %) patients without has lyzing the effects of gender, we used a hierarchical regression procedure to control for possible effects of subject age, education, age at onset of dementia symptoms, dementia duration, and family history of dementia. significant gender effects were found for the verbal task ( p < . ) (mean boston naming test score of . for women and . for men), but not for the drawing task. we conclude that verbal abilities are more severely affected in women than in men with ad, a difference that may in part reflect premorbid gender-associated differences in cerebral hemispheric organization. hemispatial placement is known to affect line bisection in patients with neglect. whereas placing stimuli in neglected space increases bisection error, placing stimuli in nonneglected space attenuates error. the effects of hemispatial placement on line bisection were examined in patients with chronic neglect (over months after stroke). all patients had large (frontotemporoparietal), unilateral, right-hemisphere lesions. each patient bisected lines of different lengths ( , , , and cm) in hemispatial conditions ( cm left of midline, midline, and cm right of midline). like previous reports, when patients bisected lines in left hemispace, a consistent ( / trials) left-sided neglect was observed ( . cm). however, when lines were bisected in center space, misbisections occurred on either side of the midline; and, unlike previous studies, when lines were bisected in right hemispace, a consistent ( / trials) right-sided neglect was observed ( . cm). the magnitude and directional consistency of line bisection errors were significant. neither visual field defects nor limitations in reaching accounted for the results. recovery in chronic neglect may involve a realignment of limited attentional resources favoring the body's midline. consequently, performance in both hemispatial fields can be biased toward midline, resulting in neglect of opposite directions. despite agreement that depression is the most common neuropsychiatric symptom associated with multiple sclerosis (ms), many aspects of this emotional change are unclear. one of the more controversial issues concerns the relationship between severity of ms and depression. this relationship is used to evaluate whether depression is an integral or reactive symptom of ms. examination of this relationship is complicated by the presumed overlap between somatic features of depressive and neurological symptoms in ms. to clarify this situation, we examined the relationship between severity of ms and categories of depressive symptoms using the beck depression inventory (bdi). eighty-nine patients and normal controls were examined. for certain comparisons, patients were classified as mild (extended disability status scale of - ) or moderatelsevere ( ) ( ) ( ) ( ) ( ) ( ) . results indicated that total bdi scores and the depressive symptom categories (mood, self-reproach, vegetative, and somatic features) were elevated in patients with ms, but the extent of these elevations was not related to severity of disease. these results suggest that depression in ms is not a simple reaction to physical disability. furthermore, clinical examination of depressive symptoms is straightforward and not confounded by severity of ms. neurological involvement in wegener's granulomatosis was studied in consecutive patients diagnosed at the mayo clinic. one hundred and nine patients ( %) had neurological involvement. peripheral neuropathy was seen in ( . %), cranial neuropathy in , external ophthalmoplegia in , cerebrovascular events in , seizures in , and miscellaneous involvement in . the mean age and sex ratio did not differ in those with or without neurological involvement. among the patients with peripheral neuropathy, had multiple mononeuropathy, had distal symmetric polyneuropathy, and had unclassified peripheral neuropathy. multiple mononeuropathy was one of the major presenting symptoms in patients. kidney involvement was significantly higher in the patients with peripheral neuropathy compared to those without it (p < . ). among the cranial nerves, the second, sixth, and seventh nerves were affected most frequently. multiple cranial nerves were affected in patients. unusual neurological manifestations among the miscellaneous group included spastic paraparesis, temporal arteritis, homer's syndrome, and papilledema. this is the first comprehensive study on the frequency and distribution of neurological involvement in wegener's granulomatosis. chronic inflammatory demyelinating polyneuropathy: a double-blind placebo-controlled crossover study treatment with high-dose intravenous human immunoglobulin (ivig) has been reported to be beneficial in some patients with chronic inflammatory demyelinating polyneuropathy (cidp), yet most observations have been nonblinded. we examined the effect of ivig therapy in patients ( men, women) with cidp in a double-blind, placebo-controlled crossover study. disease was chronic progressive (n = ) or chronic relapsing (n = ) and of variable duration ( mo to yr). the diagnosis was confirmed by electrophysiological ( ) and nerve biopsy ( ) examinations. the trial consisted of two -day periods each. patients were randomly treated with ivig ( . mg/kg/day) or placebo on consecutive days and followed. function was assessed by a quantitative neurological disability score, functional grade, grip strength measurement, and electrophysiological examinations at the beginning and end of each treatment period. with ivig therapy, significant improvement was documented in / patients (improvement in neurological disability score mean key: cord- -lp l authors: leal, laura; lauriere, mathieu; lehalle, charles-albert title: learning a functional control for high-frequency finance date: - - journal: nan doi: nan sha: doc_id: cord_uid: lp l we use a deep neural network to generate controllers for optimal trading on high frequency data. for the first time, a neural network learns the mapping between the preferences of the trader, i.e. risk aversion parameters, and the optimal controls. an important challenge in learning this mapping is that in intraday trading, trader's actions influence price dynamics in closed loop via the market impact. the exploration--exploitation tradeoff generated by the efficient execution is addressed by tuning the trader's preferences to ensure long enough trajectories are produced during the learning phase. the issue of scarcity of financial data is solved by transfer learning: the neural network is first trained on trajectories generated thanks to a monte-carlo scheme, leading to a good initialization before training on historical trajectories. moreover, to answer to genuine requests of financial regulators on the explainability of machine learning generated controls, we project the obtained"blackbox controls"on the space usually spanned by the closed-form solution of the stylized optimal trading problem, leading to a transparent structure. for more realistic loss functions that have no closed-form solution, we show that the average distance between the generated controls and their explainable version remains small. this opens the door to the acceptance of ml-generated controls by financial regulators. financial mathematics are using stochastic control to ensure that market participants are operating as intermediaries and not as unilateral risk takers: investment banks have to design risk replicating strategies, systemic banks have to ensure they have plans in case of fire sales triggered by economic uncertainty, asset managers have to balance risk and returns in their portfolios and brokers have to be sure investors' large buy or sale orders are executed without distorting the prices. the latter took a primary role in post- markets since participants understood preservation of liquidity is of primary importance. our paper addresses this last case, belonging to the academic field of optimal trading, initially introduced by [ ] and [ ] , and then extended in a lot of ways, from sophisticated stochastic control [ ] to gaussianquadratic approximations allowing to obtain closed-form solutions like [ ] or [ ] , or under a self-financing equation context in [ ] . learning was introduced in this field either via on-line stochastic approximation [ ] or in the context of games with partial information [ ] . more recently, reinforcement learning (rl) approaches have been proposed, like in [ ] to face the high dimensionality of optimal trading of portfolios, or in [ ] to adapt the controls in an online way; see also [ ] for an overview. for optimal control problems in continuous time, the traditional approach starts with the cost function and derives through dynamic programming an hamilton-jacobi-bellman (hjb) equation involving the optimal control and the value function (that is the optimum of the q-function in rl). this partial differential equation (pde) can be explicitly written only once the dynamics to be controlled are stylized enough. when it does not have a closed form solution, this pde can be solved either by a deterministic method (like a finite difference scheme) that is limited by the dimension of the problem, or by rl approaches, like in [ ] for optimal trading or in [ ] for deep hedging. more generally, deep learning techniques for pdes and backward stochastic differential equations (bsdes) have recently attracted a lot of interest [ , , , , ] and found numerous applications such as price formation [ ] , option pricing [ ] or financial systems with a continuum of agents [ , , ] . in this paper, we propose to skip most of the previous steps: we directly learn the optimal control on the discretized version of the dynamics and of the cost function that are usually giving birth to the hjb, but we no longer have to derive. this approach, also used e.g. in [ , ] for optimal control, gives us the freedom to address dynamics for which deriving explicitly a pde is not possible, and to learn directly on trajectories coming from real data. in optimal trading, the control has a feedback loop with the dynamics via price impact: the faster you trade, the more you move the price in a detrimental way [ ] . in our setup the controller sets the trading speed of an algorithm in charge of buying or selling a large amount of stock shares, solving the trade-off between trading fast to get rid of the exposure to uncertainty of future prices and trading slow to not pay too much in market impact. for numerical applications we use high frequency (hf) data from the toronto exchange (tsx) on stocks over two years, each of them generating around , trades (i.e. data points) per day. we compare different controls: controls generated by a widely known stylized version of the problem that has a closed form formula [ ] , controls learned on simulated dynamics whose parameters are estimated on the data in different ways (with or without stationary assumptions), and controls learned on real data. for the latter case, we transfer the learning from simulated data to real data, to make sure we start with a good initialization of the controller. in our setup, a controller maps the state space describing price dynamics and the trader's inventory to a trading speed that leads to an updated version of the state, and so on until the end of the trading day. the same controller is used at every decision step, which corresponds to one decision every minutes. when the controller is a neural net, if is used multiple times before the loss function can be computed and improved via back propagation, in the spirit of [ ] . our first main contribution is that we not only train a neural net for given values of the end-user's preferences, but we also train a neural net having two more inputs that are the trader's preferences (i.e. risk aversion parameters), so that this neural net is learning the mapping between the preferences (i.e. hyper-parameters of the control setup) and the optimal controls. to the best of our knowledge, it is the first time that a neural net is performing this kind of "functional learning" for optimal trading: the neural net learns the optimal control for a range of cost functions that are parametrized by the risk preferences. in the paper we call it a "multi-preferences neural net". the second major contribution of this paper is the way we compare the generated controls in a meaningful way, paving the way to methods which satisfy the request of financial regulators about the explainability of learned controls. we start by the functional space of controls spanned by the closed-form solution of the stylized problem: they are non-linear in the remaining time to trade and affine in the remaining quantity to trade (see [ ] for a description of the relationship between the optimal controls and the space generated by the h (t) and h (t) defined later in the paper). hence we project the learned controls on an affine basis of functions, for each slice of remaining time to trade t − t. doing so, we provide evidence of the distance between the black-box controls learned by a neural network, and the ones with which the end-users and regulators are familiar. we can compare the controls in this functional space, and we know, thanks to the r of the linear regressions, the average distance between this very transparent representation of the controls and the ones generated by the neural controllers. in practice, we show that when the loss function is mainly quadratic, the learned controls, even when they are trained on real data, are spanning roughly the same functional space as the closed-form ones. end-users and regulators can consider them with the same monitoring tools and analytics than more standard (but sub-optimal) controls. when the loss function is more realistic but not quadratic anymore, taking into account the mean reverting nature of intraday price dynamics [ ] , the generated controls may or may not belong to the same functional space. the structure of the paper is as follows: section presents the optimal execution problem setup, focusing on the loss function and the closed-form formula associated with a stylized version of the problem that we will use as a benchmark. it also introduces the architecture of the neural networks we use and our learning strategies. section describes the dataset, stylized facts of intraday price dynamics that should be taken into account, and motivates learning in a data-driven environment. section presents the numerical results and our way to tackle explainability of the generated controls. conclusions and perspectives are provided in section . the optimal execution model optimal trading is dealing with an agent who would like to execute a large buy or sell order in the market before a time horizon t . here, their control is a trading speed ν t ∈ r. the center of the problem is to find the balance between trading too fast (and hence move the price a detrimental way and pay trading costs, as modelled here in ( ) and ( )) and trading too slow (and being exposed to not finishing the order and be exposed to uncertainty of future prices, reflected in ( ) and ( )). they maximize their wealth while taking into account a running cost and a final cost of holding inventory, and they are constrained by the dynamics of the system, which is described by the evolution of the price s t , their inventory q t , and their wealth x t . in the market, the price process for the asset they want to trade evolves according to where α t > is a parameter that accounts for the drift, ∆t is the size of a time step, σ > is a constant volatility term and ∼ n ( , ) is a noise. the state of the investor at time t is described by the tuple (t − t, q t , x t ), with q t being their inventory, and x t being their wealth at time t. to isolate the inventory execution problem from other portfolio considerations, the wealth of the agent at time is considered to be , i.e. x = . suppose the agent is buying inventory (the selling problem is symmetric). then, q < and the trading speed ν will be mostly positive throughout the trajectory. the evolution of the inventory can be written as: the wealth of the investor evolves according to where κ > is a constant, and the term κ · ν t represents the temporary market impact from trading at time t. as we can see, this is a linear function of ν t that acts as an increment to the mid-price s t . it can also be seen as the cost of "crossing the spread", or a transaction cost. the cost function of the investor fits the framework considered e.g. in [ ] . the agent seeks to maximize over the trading strategy ν the reward function given by: with γ > . we mostly focus on the standard case γ = , but we will also consider the case where γ = / , which better takes into account the sub-diffusive nature of intraday price dynamics, see [ ] . x t , s t , and q t are the random variables parameterizing the terminal value of the stochastic processes described in the dynamics ( )- ( ) . a > and φ > are constants representing the risk aversion of the agent. a penalizes holding inventory at the end of the time period, and φ penalizes holding inventory throughout the trading day. together, they parametrize the control of the optimal execution model and stand for the agent's preferences. the trading agent wants to find the optimal control ν for the cost ( ) subject to the dynamics ( )-( ). in the sequel, we solve this problem using a neural network approximation for the optimal control. as a benchmark for comparison, we will use closed-form solution for the corresponding continuous time problem, which is derived through a pde approach. the closed-form solution of the pde is well-defined only when the data satisfies suitable assumptions. the neural net learns the control ν t directly from the data, while finding a closed-form solution for the pde is not always possible. it can be shown, see [ ] , that the optimal control ν * is obtained as a linear function of the inventory, which can be written explicitly as: where h and h are the solutions of a system of ordinary differential equations (odes). the neural network setup. the neural network implementation we propose precludes all the usual derivations in the [ ] framework. we no longer need to find the pde corresponding to the stochastic optimal control problem, we no longer need to break it down into odes and solve the ode system, and we can try to directly approximate the optimal control. the deep neural network approximation looks for a control minimizing the agent's objective function ( ) while being constrained by the dynamics ( )-( ), without any other derivations. we define one single neural network f θ (·) to be trained for all the time steps. each iteration of the stochastic gradient descent (sgd) proceeds as follows. starting from an initial point (s , x , q ), we simulate a trajectory using the control ν t = f θ (t, q t ). based on this trajectory, we compute the gradient of associated cost with respect to the neural network's parameters θ. finally, the network's parameters are updated based on this gradient. in our implementation, the learning rate is updated using adaptive moment estimation (adam) [ ] , which is well suited for situations with a large amount of data, and also for non-stationary, noisy problems like the one under consideration. in the monte-carlo simulation mode, we generate random increments of the brownian motion through the term σ √ ∆t , where ∼ n ( , ) comes from the standard gaussian distribution. using the gaussian increments, along with the ν t obtained from the neural network, and the three process updates (∆s t , ∆x t , ∆q t ), we can compute the new state variables in discrete time: the new state variable q t+ , along with the time variable, is then going to serve as input to help us learn the control at the next time step. this cycle continues until we have reached the time horizon t . since we are using the same neural network f θ for all the time steps, we expect it to learn according to both time and inventory. neural network architecture and parameters. the architecture consists of three dense hidden layers containing five nodes each, using the hyperbolic tangent as activation function. each hidden layer has a dropout rate of . . we add one last layer, without activation function, that returns the output. the learning rate is η = e − ; the mini-batch size is ; the tile size is ; and the number of sdg iterations is , . every sgd iterations, we perform a validation step in order to check the generalization error, instead of evaluating just the mini-batch error. in our implementation, we used tensorflow. although the neural network's basic architecture is not very deep, each sgd step involves t loops over the same neural network. this is because of the closed loop which the problem entails. the number of layers is thus artificially multiplied by the number of time steps considered. for the inputs, we have two setups. in the basic setup (as discussed above), the input is the pair (t, q t ). in the second case, which we call "multi-preferences neural network ", the input is the tuple (t, q t , a, φ) and the neural netowrk learns to minimize the j a,φ (·) cost functions for all (a, φ). each time we need to solve a given system, we set a and φ to the desired value in the multi-preferences network and we do not need to relearn anything to obtain the optimal controls. for both neural nets, the output is the speed of trade ν t for each time step t, intercalating with the variable updates, thus learning a controller for the full length of the trading day. in order to learn from historical data, we first train the network on data simulated by monte carlo and then perform transfer learning on real data. we would like to compare the control obtained using the pde solution with the control obtained using the neural network approximation. as stated in equation ( ), the optimal control can be written as an affine function of the inventory q t at any point in time, for t ∈ [ , t ]. the shape of this closed-form optimal control belongs to the manifold of functions of t and q spanned by [ , t ]×r (t, q) → h (t)/( κ)+(α+h (t))/( κ)·q ∈ r, where the h i (t) are non-linear smooth functions of t. to provide explainability of our neural controller we project its effective controls on this manifold. we naturally obtain two non-linear functionsh (t) anh (t) and a r (t) measuring the distance between the effective control and the projected one at each time step t. procedure of projection on the "closed-form manifold". for each t, we form a database of all the learned controls ν t mapped by the neural net to the remaining quantity q t . it enables us to project this ν t on q t using an ordinary least squares (ols) regression. the coefficients β (t) and β (t) of this regression ν t = β (t) + β (t)q t + ε t can be easily inverted to givẽ for each t ∈ [ , t ]. the r (t) of each projection associated to t quantifies the distance between the effective neural "black box" control and the explained oneν t :=h (t)/( κ) + (α +h (t))/( κ) · q t . the curve of r (t) represents how much of the non-linear functional can be projected onto the manifold of closed-form controls at each t. in practice we perform t = ols projections to obtain the curves of h , h and r (see figure ) which provide explainability of our learned controls. users' preferences and exploration-exploitation issues the rate at which the agent executes the whole inventory strongly depends on the agent's preferences a and φ. when they are both large, the optimal control tends to finish to trade earlier than t , the end of the trading day. because of that, in such a regime the tile size (ts) stands for how many samples of the inventory we combine with each sampled pair. for ts = , each pair where j is the tile index. this is useful when using real data, because we can generate more scenarios than we would ordinarily be able to. it will be very difficult for the neural net to learn anything around the end of the time interval since it will no more explore its interactions with price dynamics to be able to emulate a better control. for the case of the multi-preferences controller with inputs, including a and φ, taking (a, φ) in a certain range ensures that the neural net will witness long enough trajectories to exploit them. to this end, we took profit of the closed-form solution of the stylized dynamics and scanned the duration of trajectories for each pair (a, φ). in order to avoid the exploration -exploitation trade-off, we restricted the domain to values of φ smaller or equal to . , and values of a smaller or equal to . . this enabled the regression to learn the functional produced by the neural network. there will be some inventory left to execute at the end of the trading day for these parameters. for the pair (a, φ) = ( . , . ), we have less than % of the initial inventory left to execute, yet it is enough for us to estimate the regression accurately. optimal execution with data we are using ticker data for trades and quotes for equities traded on the toronto stock exchange for the period that ranges from jan/ until dec/ , which results in trading days. both trades and quotes are available for level i (no level ii available) for stocks (see table ). they have a diverse range of industries, and daily traded volume. our method can be directly used for all the stocks, either individually or simultaneously. table we partition both trade and quote files into smaller pickled files organized by date. we merge them day by day to calculate other interesting variables, such as: trade sign, volume weighted average price (vwap), and the bid-ask spread at time of trade. we drop odd-lots from the dataset, as they in fact belong to a separate order book. moreover, we only keep the quotes that precede a trade, or a sequence of trades. it saves us memory usage and reduces processing times. the trade data, including the trade sign variable, is further processed into five minute bins. taq data is extremely asynchronous and a situation might happen when there is not enough data in a given time interval. to avoid this problem, we aggregate the data into bins and focus our analysis on liquid stocks. this bin size is big enough for us to have data at each bin, and small enough to allow the agent to adjust the trading speed several times throughout the day. since the market is open from : until : , five-minute bin intervals, we have time steps, which gives us bins for which to estimate the control on each trading day. we have . % of bins containing data (see table ). the stylized dynamics allowing a pde formulation of the problem and a closed-form solution of the control make a lot of assumptions of the randomness of price increments, market impact and trading costs. typically they assume independent and normally distributed, stationary, non-correlated returns, with no seasonality. however, it is not the case for financial time series. see [ , , ] for some interesting discussions. figure shows the specific characteristics of financial data for the stock mru. the left plot is a qq-plot, which tells us that the stock returns have a heavy tailed distribution. the same is confirmed for all stocks in table : we observe high kurtosis for all the distributions of stock returns(which is a common metric for saying that a distribution has heavy tails with respect to a gaussian distribution). the middle plot presents the five-minute lag auto-correlation profile. for this stock, we can see a mean-reversion tendency for five minutes and one hour. the first lag of the five-minute auto-correlation is shown for all stocks in table . the right plot shows intra-day seasonality profiles: the bid-ask spread (blue, dashed curve), and the intra-day volume curve (full, gray curve). learning the mapping to the optimal control in a closed loop as mentioned in section . , the first step is to compare the output of our neural network model to the pde solution, both using monte carlo simulated data. the second step is to understand how seasonality in the data might affect the training and the output. finally, we continue our learning process on real data, in order to learn its peculiarities in terms of heavy tails, seasonality and auto-correlation. the results and our benchmark are summarized in figure . as stated in equation ( ), the optimal control for the stylized dynamics and the pde is linear in the inventory, hence the associated r is obviously for any t. for the different neural nets, we perform the projections on the (h , h ) manifold and keep track of the r curve. during the learning, it is important that the neural network can observe full trajectories. when the risk aversion parameters are very restrictive, the closed-form solution and neural net trades so fast that the order is fully executed (i.e. the control stops) far before t = t . because of that it is impossible to learn after this stopping time corresponding to q t = . our workaround for this exploration -exploitation issue has been to use the closed-form solution to select a range of (a, φ) allowing the neural net to generate enough trajectories to observe the dynamics up to t = t . in figure in this figure , we mix closed-form controls on the stylized dynamics, neural controls on the same dynamics (using monte carlo simulations), neural controls in more realistic simulations (with an intraday seasonality) and ultimately on real data. we also superpose results for neural networks trained only on this regime of preferences and the controls of the multi-preference neural network, that is trained for once and then generates controls for any pair (a, φ) of preferences. the top panels represent the two components of the projection of the control on the "closed-form manifold", to enable comparison. the r (t) curves (bottom-left panel) are flat at for the closed form formula (since % of the control belongs to this manifold), whereas it can vary for the other controls (more cases are available in the appendix). thanks to this projection, it is straightforward to compare the behaviour of all the controls. the r (t) curves provide evidence that in this regime the neural controls are almost % explained by the closed-form manifold. it does not say that they are similar to the closed-form solutions of the stylized problem, but that they are linear in q t (but not in t) for each time steps t. for non quadratic costs (we took γ = / since it is a realistic case [ ] ) we take (a, φ) = ( . , . ) to be in a comparable regime as the other controls. the r (t) curve shows that for these parameters, the learned control remains extremely close to the closed-form manifold. this kind of evidence can allow risk departments of banks and regulators to get more comfortable with this learned control. main differences between the learned controls. the different learning contexts are primarily influencing h (t), that is a shift in the trading speed. this is an important result by itself in optimal trading: going from stylized dynamics, to simulations with intraday seasonality, and then to real price dynamics does not change that much the multiplicative term of q t , but shifts the trading speed. this has been already observed in the context of game theoretical frameworks [ ] . when the seasonality is added in the simulation (dotted green curve) the deformation of the control exhibits oscillations that most probably stem from the seasonalities of the right panel of figure . the shift learned on real price dynamics (dashed orange) amplifies some oscillations that are now less smooth; this is probably due to autocorrelations of price innovations. moreover figure shows that the "functional learning" worked: the mono-preference neural network trained only on this (a, φ) and the multi-preferences one have similar curves. in this paper, we succeed in using a neural network to learn the mapping between end-user preferences and the optimal trading speed, allowing to buy or sell a large number of shares or contracts on financial markets. prior to this work, various proposals have been made up to now but the learned controls have always been specialized for a given set of preferences. here, our multi-preferences neural network learns the solution of a class of dynamical systems. note that optimal execution dynamics are reacting to the applied control, via the price impact of the buying or selling pressure. the neural network hence has to learn this feedback. our approach uses a deep neural network, whose inputs are user preferences and the state of the optimization problem that change after each decision. the loss function can only be computed at the end of the trading day, once the neural controller has been used times. the backpropagation hence takes place across these steps. we faced some exploration -exploitation issues and solved it by choosing suitable the range of users' preferences to ensure that long enough trajectories are observed during the learning. our setup leverages on transfer learning, starting on simulated data before switching to historical data. since we want to understand how the learned controls are different from the closed-form solution on a stylized model that are largely used by practitioners, we learn on different versions of simulated data: from a model corresponding to the stylized one to one incorporating non-stationarities, and then to real data. to ensure the explainability of our learned controls, we introduce a projection method on the functional space spanned by the closed-form formula. it allows us to show that most of the learned controls belong to this manifold, and that the source of adaptation to realistic dynamics focuses on a "shift term" h in the control. the most noticeable adaptation of h exhibits slow oscillations that are most probably reflecting the seasonalities of intraday price dynamics. we then introduce a version of the loss function that reflects more the reality of intraday price dynamics (that are sub-diffusive). despite the fact that the associated hjb has no closed form solution, we manage to learn its associated optimal control and show, using our projection technique, that it almost belongs to the same manifold. this approach delivers explainability of learned controls and can probably be extended to contexts other than optimal trading. it should help regulators to have a high level of trust in the learned controls, that are often considered as "black boxes": our proposal exposes the fraction of the controls that belongs to the manifold practitioners and regulators are familiar with, allowing them to perform the usual "stress tests" on it, and quantifies this fraction as a r curve that is easy to interpret. explainability of learned control for financial markets. attempts of using machine learning for financial markets are booming the last years. it spans a wide spectrum of applications: from client profiling to nowcasting using databases of texts and satellite images. one area where the acceptance of ml goes slowly is the automation of hedging strategies. these strategies are part of the functioning of financial markets in the post -crisis environment, because genuine regulations demand to market participants to compute and compensate their exposure to as much risk scenarios as possible. moreover and for obvious reasons, regulators ask for a better understanding of algorithms that look like "black boxes" before allowing the use of ml in production to improve the efficiency of hedging. it is related to the very well known topic of "explainability of ai ". optimal trading is addressing part of these hedging strategies, since a framework like the one used in this paper corresponds to an "large investor", typically a pension fund, who decided to rebalance its portfolio for good reasons (think about the market moves following the spread of covid- : the optimal response of pension funds is clearly to rebalance their portfolio to get less exposure to factors like airplane companies, to prevent their pensioners to suffer from losses in the coming year), but they have to do it at a slow enough pace to not push the price too much at their own detriment [ ] . the reason for the big covid- drop of markets followed by a rebound days later is due to non optimal trading strategies: the institutions sold too fast, paying too much for their deleveraging and perturbing the prices with the downward pressure of their too fast sells. being able to adapt the trading strategies to realistic features of intraday price dynamics (like seasonality and auto-correlations) decreases both the trading costs for large institutions like pension funds (and consequently goes back to their pensioners) and is profitable to the public price formation, that is otherwise more blurred and does not reflects the "fair value" of traded instruments. in the introduction, we cite papers on improving optimal trading, via reinforcement learning or directly using deep learning. they do not attempt to provide explainable version of the obtained controls. in this paper we do it in a very practical way that is compliant with the practices of the financial industry: we project the learned controls on the manifold spanning the controls currently in use by brokers and asset managers, and measure the distance between the obtained projection and the learned controls (via a r curve that is straightforward to understand). doing this: we provide evidences that the learned controls that we generated are largely contained in this regular manifold, and we allow practitioners and regulators to apply their usual stress testing and certification practices to the projected controls. it is the first proposal in this direction, and we hope that it will propagate in other areas of finance, like deep hedging. it may also be used in other industries when the regulatory demand in explainability is high. "functional learning" for control. the second "broader impact" of this paper is what we called "functional learning" in the scope optimal trading with the setup we call multi-preferences neural network. in other applications of optimal control, the loss function is fixed because it correspond to one universal use of the controlled object. on financial markets the loss function is parametrized by hyperparameters describing the risk aversion of the end-user. in game theory versions of the optimal trading framework, they are usually called "agent preferences" [ ] . it is like allowing the driver of an autonomous car to choose the "driving style" she or he wants to use: far below the speed limit (may be for low carbon emission reasons) or just below, or a "sport mode", etc. on financial markets, mainly because on the one hand models of price impact have confidence intervals and on the other hand asset owners can have a lot of reasons to rebalance their portfolio (think about an australian pension fund that has to sell fast enough to face the exceptional pensioners' redemption authorized by the government following the covid- crisis; it is clear that the selling speed has to be fast enough to give the money to pensioners), these parameters are chosen at the start of trading (t = in our framework) by the end-user. today a stylized version of the control problem is numerically solved on the fly (either in closed-form, either by a numerical scheme) for the chosen preferences, but the dynamics have to be stylized, hence simplified, enough to allow to solve this very quickly. learning the optimal control each time an end-user choose new preferences is not fast enough. in this paper the neural net is learning to solve the optimal control problem for any preference; in fact it learns the mapping between the preferences and the optimal solution. it effectively learns to solve a whole parametrized family of hamilton-jacobi-bellman equations. hence it can provide the optimal trading strategy to a given choice of preferences in a flash. keep in mind this optimal strategy is not one number but it is itself a mapping between the state space of the control problem and the optimal trading speed to be applied. it had chances to work because it is straightforward to check (in the continuous setting) that the infinitedimensional optimal control strategy is a smooth function of the two-dimensional vector of preferences (a, φ), hence we ask to the neural network to interpolate high dimensional functions in this -dimensional grid. nevertheless it is far from easy (that's probably why other attempts did not succeed by now). in the paper, we refer to what we suspect to be the main difficulty under the name of "exploration -exploitation issue". for some pairs of user preferences, the optimal trading speed is so fast that the full order is bought or sold far before t , and hence the neural network cannot learn from the feedback of its control on the dynamics, simply because it stopped to interact with them. to counter this we used the closed-form formula of the stylized problem to define a domain of user preferences that is compatible with our t , it helped the convergence of the "functional learning". for the sake of completeness, we recall how the benchmark solution is obtained; see [ ] for more details. the continuous form of the problem defined in equations ( ) to ( ), when γ = , can be characterized by the value (we drop the subscripts a and φ to alleviate the notations) where v is the value function, defined as: from dynamic programming, we obtain that the value function v satisfies the following hamilton-jacobi-bellman (hjb): for t ∈ [ , t ), x, s, q ∈ r, with terminal condition v (t, x, s, q) = x + q(s − aq). if we use the ansatz v (t, x, s, q) = x + qs + u(t, q), with u of the form u(t, q) = h (t) + h (t)q + h (t) q , the optimal control resulting from solving this problem can be written as: hence, h (t) and h (t) act over the control by influencing either the intercept of an affine function of the inventory, in the case of h (t), or its slope, in the case of h (t). from the hjb equation, these coefficients are characterized by the following system of ordinary differential equations (odes): with terminal conditions: in this section, we provide more details on the implementation of the method based on neural network approximation. for the neural network, we used a fully connected architecture, with three hidden layers, five nodes each. one forward step in this setup is described by figure , while one round of the sgd is represented by figure . the same neural network learns from the state variables obtained in the previous step. it thus learns the influence of its own output through many time steps. we have experimented with using one neural network per time step. however, this method implies more memory usage, and did not provide any clear advantage with respect to the one presented here. from figures and , we can clearly see the idea that the control influences the dynamics. we are, in fact, optimizing in a closed-loop learning environment, where the trader's actions have both permanent and temporary market impact on the price. the mini-batch size we used, namely , is relatively small. while papers like [ ] defend the use of larger mini-batch sizes to take advantage of parallelism, smaller mini-batch sizes such as the ones we are using are shown to improve accuracy in [ ] , [ ] and [ ] . figure : structure of the state variables' simulation -one step permanent market impact let s be the space of stocks; and d be the space of trading days. if we take five-minute intervals (indicated by the superscript notation), we can write equation ( ) for each stock s ∈ s, for each day d ∈ d, and for each five-minute bin indexed by t as: where the subscripts s, d, t respectively indicate the stock, the date, and the five-minute interval to which the variables refer, and ∆t = min, by construction. we have α s,t independent of d, which assumes that for any given day the permanent market impact multiplier the agent may have on the price of a particular stock s, for a given time bin t is the same. and we have σ s independent of the day d and time bin t, which means the volatility related to the noise term is constant for a given stock. as an empirical proxy for the theoretical value ν min s,d,t representing the net order flow, we use the order flow imbalance observed in the data: imb min s,d,t . we define this quantity as: where v buy s,d,t is the volume of a buy trade, and v sell s,d,t is the volume of a sell trade, and we aggregate their net amounts over five minute bins. however, since we are estimating the permanent market impact parameter α using data from different stocks, and we would like to perform a single regression for all of them, we re-scale the data used in the estimation. in order to make the data from different stocks comparable, we do the following: . divide the trade price difference (∆s min s,d,t ) by the average bin spread over all days for the given stock, also calculated in its respective five minute bin: where ψ min s,d,t is the bid-ask spread for a given ( . where total volume min s,d,t stands for the total traded volume for both buys and sells for a given (stock, date, bin) tuple. this ensures that both the volume and the price are normalized to quantities that can be compared. each (s, d, t) now gives rise to one data point, and we can thus run the following regression instead, using comparable variables, to findᾱ: whereᾱ is the new slope parameter we would like to estimate, and¯ min s,d,t is the normalized version of the residual we had in equation ( ) . in order to useᾱ in a realistic way, we de-normalize the regression equation for each stock by doing: wherev s,t is the average bin volume for stock s at bin the time bin t,ψ s,t is the average bin bid-ask spread for the same pair (s, t). the derivation for the temporary market impact κ uses equation ( ), and follows similar steps. in section . , we discussed the exploration -exploitation trade-off we encountered when projecting the neural network controller onto the manifold of closed-form solutions when γ = . in figure , we compare the number of time steps it takes the controller to trade at least % of the initial inventory in the market. rows stand for values of the risk parameter a ∈ { . , . , . }, while columns represent the risk parameter φ ∈ { . , . , . , . , e − , e − }. when the trader's preferences over risk aversion gives them incentive to trade faster, the inventory is executed sooner. the controller is 'exploiting' the available trading choices, and the projection on the closed-form manifold becomes harder to estimate. on the other hand, if the pair (a, φ) defines less restrictive preferences towards execution, there will be some (although not a lot of) inventory left at the end of the trading day that allows us to learn the projection accurately. when γ = / , the projection on the "shift term" h is larger than usual, and the absolute value of "proportional term" h is very close to zero when using the same scale as the h when γ = (it is increasing with time, that is the reverse of usual when in its own scale). nevertheless, it is compensated by the part of the control that is outside of this space, as indicated by the r : at the start of the trading process % of the control is explained by the "closed-form manifold"; it then decreases to %, meaning that the learned control takes part fully out of this manifold; but it then increases back to % at the end of the process. it seems that the best way to finish the trading is already well captured by the closed-form manifold. as we observe from the control plot in the bottom-right of figure , the parameter pair (a, φ) = ( . , . ) does not enforce trading speeds as fast as the pair (a, φ) = ( . , . ) when the loss function is sub-diffusive. keeping the same preferences as before is thus making the trader less aggressive in a sub-diffusive environment. in order to have the same behavior in terms of control, they would need to behave in a more risk averse manner. average control process (nu) figure : explainable parts of the control: h (t), h (t); fraction of the explained control r (t), and average control given it is positive e(ν(t)|ν(t) > ) for different controllers, for the case γ = / . optimal execution of portfolio transactions market impacts and the life cycle of investors orders optimal control of execution costs tails, fears, and risk premia optimal control of trading algorithms: a general impulse control approach trades, quotes and prices: financial markets under the microscope deep hedging trading volume and serial correlation in stock returns mean field game of controls and an application to trade crowding convergence analysis of machine learning algorithms for the numerical solution of mean field control and games: i-the ergodic case convergence analysis of machine learning algorithms for the numerical solution of mean field control and games: ii-the finite horizon case the self-financing equation in limit order book markets incorporating order-flow into optimal execution algorithmic and high-frequency trading reinforcement learning in economics and finance large scale distributed deep networks deep learning-based numerical methods for high-dimensional parabolic partial differential equations and backward stochastic differential equations deep learning methods for mean field control problems with delay sensitivity analysis using itô-malliavin calculus and martingales, and application to stochastic optimal control deep reinforcement learning for market making in corporate bonds: beating the curse of dimensionality accelerated share repurchase and other buyback programs: what neural networks can bring deep learning approximation for stochastic control problems solving high-dimensional partial differential equations using deep learning some machine learning schemes for high-dimensional nonlinear pdes on large-batch training for deep learning: generalization gap and sharp minima adam: a method for stochastic optimization optimal starting times, stopping times and risk measures for algorithmic trading market microstructure in practice optimal posting price of limit orders: learning by trading piecewise affine neural networks and nonlinear control pricing options and computing implied volatilities using neural networks. risks revisiting small batch training for deep neural networks improving reinforcement learning algorithms: towards optimal learning rate policies physics-informed neural networks: a deep learning framework for solving forward and inverse problems involving nonlinear partial differential equations universal features of price formation in financial markets: perspectives from deep learning dgm: a deep learning algorithm for solving partial differential equations the general inefficiency of batch training for gradient descent learning the authors are extremely grateful for the fruitful discussions with professor rené carmona, who was also very kind to provide the data we are using. the work of l. leal and m. laurière was supported by nsf dms- and aro w nf- - - . key: cord- -sb mjhzm authors: moore, stephen e.; okyere, eric title: controlling the transmission dynamics of covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: sb mjhzm the outbreak of covid- caused by sars-cov- in wuhan and other cities in china in has become a global pandemic as declared by world health organization (who) in the first quarter of . the delay in diagnosis, limited hospital resources and other treatment resources leads to rapid spread of covid- . in this article, we consider an optimal control covid- transmission model and assess the impact of some control measures that can lead to the reduction of exposed and infectious individuals in the population. we investigate three control strategies for this deadly infectious disease using personal protection, treatment with early diagnosis, treatment with delay diagnosis and spraying of virus in the environment as time-dependent control functions in our dynamical model to curb the disease spread. the recent outbreak of the deadly and highly infectious covid- disease caused by sars-cov- in wuhan and other cities in china in has become a global pandemic as declared by world health organization (who) in the first quarter of [ ] . the most vulnerable people to develop serious complications from this dangerous disease are the elderly with underlying medical problems. as at th march, , the th situation report by who indicated that the deadly covid- disease had globally infected , people with , deaths, see [ ] . the understanding of the transmission dynamics of infectious diseases has been well-studied and researched in mathematics and usually referred to as mathematical epidemiology. these mathematical models have played a major role in increasing understanding of the underlying mechanisms which influence the spread of diseases and provide guidelines as to how the spread can be controlled [ , , ] . the recent outbreak of the deadly and highly infectious covid- disease has attracted the attention of many authors who have discussed and studied the nature of the virus, its transmission dynamics and the basic reproduction number of the disease, see eg. [ , , , , , ] . recently, elsevier and springer have made open access to several literature for interested researchers [ , ] . an seir mathematical model for the transmission dynamics of covid- disease with data fitting, parameter estimations and sensitivity analysis is studied in [ ] whiles a deterministic model for covid- that captures the effect of delay diagnosis on the disease transmission has also been presented, see [ ] . in [ ] , the authors explore a statistical analysis of covid- disease data to estimate time-delay adjusted risk for death from this deadly virus in wuhan, as well as for china excluding wuhan. their study suggested that effective social distancing and movement restrictions practices can help minimise the disease transmission. a real-time forecast phenomenological model has also been designed to study the transmission pattern of covid- infectious disease, see e.g., [ ] . also, an seir-type compartmental modelling concept applied to design a data-driven epidemic model that incorporates governmental actions and individual behavioural reactions for the covid- disease outbreak in wuhan [ ] . mathematical modelling of epidemics using deterministic optimal control problems is widely explored in the literature of mathematical epidemiology. a detailed comprehensive literature of optimal control models in epidemiological modeling and numerical approximation techniques can be found in [ , ] . several works in literature reveals that epidemic models that are constructed with optimal control problems are appropriate and very useful for suggesting control strategies to curb disease spread, see, e.g., [ , , , ] . the principal purpose of this article is to present control strategies for transmission dynamics of covid- and to determine strategies that are critical even during instances of delay in diagnosis. in section , we formulate an optimal control model for covid- with four control measures. the analysis of the control model is presented in section . in section , we present the numerical results of the optimal control model. finally, we conlude in section with discussions on the control measures. in this section, we formulate an optimal control model for covid- to derive four control measures with minimal implementation cost to eradicate the disease after a defined period of time. our new epidemiological time-dependent control model is an extended and modified version of the covid- transmission dynamical model introduced in [ ] . here, we note that the population is divided into susceptible (s), self-quarantine susceptible (s q ), exposed (e), infectious with timely diagnosis (i ), infectious with delayed diagnosis (i ), hospitalized (h), recovered (r) and the viral spread in the environment (v ). following the compartmental transition diagram as shown in figure , the eightstate dynamical model describing the transmission dynamics of covid- is given by , see [ ] . where β e , β i , β i and β v denote the transmission rates from the exposed, infectious with timely diagnosis, infectious with delay diagnosis and virus in the environment to the susceptible, respectively. also f i , i = , , is the rate of virus in the environment from both the exposed and the infectious and removed at rate d v . for the rest of the model parameters, we refer the interested reader to reference [ ] for detailed descriptions. following from the system ( . ), we modified the transmission rate by reducing the factor by ( − u ), where u measures the effort of individuals to protect themselves (i.e. personal protection). the control variable u measures the treatment rate of timely diagnosed individuals whiles the u measures the treatment rate of delayed diagnosed individuals. we assume that u i and u i individuals are removed from the timely diagnosed class and delayed diagnosed class and added to the hospitalized class. the fourth control variable u measures the spraying of the environment to prevent viral release. we also assume that u v virus are removed from the environment. we further assume that individuals that recovers at any time t after hospitalization and treatment are removed from the hospitalized class to the recovered class. with regards to these assumptions, the dynamics of system ( . ) are modified into the following system of equations: where q is the rate at which susceptible (s) individuals move into self-quarantine (s q ) and q is the rate at which self-quarantined individuals become susceptible again. also, µ is the death rate and hospitalized individuals are decreased at recovery rate m. in this section, we will formulate an objective functional and present the existence of optimal control by means of pontryagin's maximum principle. given the optimal control problem ( . ), we prove the existence of control problem following [ ] and then characterizing it for optimality. the objective functional j formulates the optimization problem of identifying the most effective strategies. the overall preselected objective involves the minimization of the number of exposed, delayed diagnosed infectious individuals and the viral spread in the environment over a finite time interval [ , t ]. we define the objective functional j , as follows we aim to minimize the cost functional ( . ) which includes the number of exposed (e), infectious with delay diagnosis (i ), and the virus in the environment (v ), as well as the social costs related to the resources needed for personal protection c u , early detected treatment c u , delay detected treatment c u , and spraying of environment the objective of the control problem is to seek functions (u * (t), u * (t), u * (t), u * (t)) such that where the control set is defined as subject to the covid- model with controls ( . ) and appropriate initial conditions. in the next section, we prove the existence of an optimal control for the system ( . ) and then derive the optimality system. it is well known that pontryagin's maximum principle (pmp) is required to solve this control problem and the derivation of the necessary conditions [ , ] . the necessary conditions include the optimality solutions and the adjoint equations that an optimal control must satisfy which come from pontryagin's maximum principle [ ] . this principle converts the control model ( . ) and the objective functional ( . ) into a problem of minimizing pointwise hamiltonian function ( . ), which is formed by allowing each of the adjoint variables to correspond to each of the state variables accordingly and combining the results with the objective functional. theorem . given the objective functional j (u , u , u , u ) as in ( . ), where the control set u given by ( . ) is measurable subject to ( . ) with initial conditions given at t = , then there exists an optimal control u * = (u * (t), u * (t), u * (t), u * (t)) such that j (u * (t), u * (t), u * (t), u * (t)) := min{j (u , u , u , u ), (u , u , u , u ) ∈ u}. proof. the existence of an optimal control due to the convexity of the integrand of j with respect to the control measures u i , i = , . . . , , an a priori boundedness of the solutions of both the state and adjoint equations and the lipchitz property of the state system with respect to the state variables follows from [ ] . to find the optimal solution, we need the lagrangian (l) and hamiltonian (h) for the optimal control problem ( . ) and ( . ). the lagrangian of the control problem is given by since we want the minimal value of the lagrangian, we define the hamiltonian function for the system as where λ j , j ∈ {s, s q , e, i , i , h, r, v } are the adjoint variables. next, we apply the necessary conditions to the hamiltonian h in ( . ). theorem . given an optimal control u * := (u * , u * , u * , u * ) and a solution y * = (s * , s * q , e * , i * , i * , h * , r * , v * ) of the corresponding state system ( . ), there exists adjoint variable λ j , j ∈ {s, s q , e, i , i , h, r, v } satisfying with transversality conditions λ j (t ) = , j ∈ {s, s q , e, i , i , h, r, v }. furthermore, the control functions u * , u * , u * and u * are given by proof. the proof methodology follows similarly as presented by [ ] . in this section, we present the numerical solutions for our optimality problem using the fourth-order runge-kutta forward-backward sweep method. this numerical scheme is very efficient and has been widely used by several authors in simulating their optimal control problems [ , , [ ] [ ] [ ] . the details of this scheme can be found in the monograph [ ] . parameter values for our numerical illustrations are adapted from [ ] where the authors used data from the city of wuhan in the hubei province of china. we assume a = , a = , a = , c = , c = , c = and c = . figure below represent profiles of the optimal control functions (u , u , u , u ). in this strategy, we consider personal protection (u = ), treatment with early diagnosis (u = ), treatment with delay diagnosis (u = ) time-dependent control functions to minimise our objective functional. our main aim in this control strategy is to minimise the number of exposed (e), infectious with delay diagnosis (i ) and virus in the environment (v ). in the non-optimal control model ( . ), treatment with early diagnosis (γ = u = . ) and treatment with delay diagnosis (γ = u = ) are captured as constant controls. fig. . solution trajectories for exposed individuals with varying parameter φ = . , φ = . and φ = . . the red line represents the controlled exposed population whiles the blue line represents the uncontrolled exposed population. this strategy deals with treatment control with early diagnosis (u = ) and treatment control with delay diagnosis (u = ) to minimise our objective functional. our main aim in this control strategy is to minimise the number of exposed (e), infectious with delay diagnosis (i ) and virus in the environment (v ). in the non-optimal control model ( . ), treatment with early diagnosis (γ = u = . ) and treatment with delay diagnosis (γ = u = ) are captured as constant controls. fig. . solution trajectories for exposed individuals with varying parameter φ = . , φ = . and φ = . . the red line represents the controlled exposed population whiles the blue line represents the uncontrolled exposed population. in this strategy all the four time-dependent control functions (u = , u = , u = , u = ) proposed in this study are incorporated into the optimal control covid- model problem to minimise the objective function. in the non-optimal control model, treatment with early diagnosis (γ = u = . ) and treatment with delay diagnosis (γ = u = ) are captured as constant controls. fig. . solution trajectories for exposed individuals with varying parameter φ = . , φ = . and φ = . . the red line represents the controlled exposed population whiles the blue line represents the uncontrolled exposed population. in this subsection, solution trajectories for the number of exposed, infectious with delay diagnosis and virus in the environment for all the three control strategies are numerically compared with that of the non-optimal control model. our numerical results suggest that, if people can adhere to effective personal protection practices such as the use of hand sanitizers, washing of hands regularly and social distancing, there will less infections in the population. from our results, we can further argue that, effective spraying of the environment and early diagnosis of infected or infectious individuals and treatment can help reduce of the number of covid- infections significantly. in this article, we presented control strategies to the transmission dynamics of covid- . the control set including personal protection, treatment when individuals are early diagnosed, treatment when individuals are delay diagnosed, effective spraying of the environment and cleaning possible infected surfaces can help reduce the quantity of the virus. the numerical simulations reveals that optimal control strategies can yield significant reduction of the number of covid- exposed and infectious or infected individuals in the population. by instituting control strategies, we realise that the number of days required for the virus to be eliminated from the system is significantly reduced as compared to when there is no control strategy. the numerical illustrations also shows that by increasing φ i.e. improving the diagnostic resources, and increasing γ i.e. improving the diagnostic efficiency, we can control significantly the number of new confirmed cases, new infections and thus can reduce the transmission risk. from all the three control strategies considered in this study, we realised that the third strategy which captures all the four time-dependent control functions yields better results. modelling the effects of heavy alcohol consumption on the transmission dynamics of gonorrhea with optimal control mathematical epidemiology: past, present, and future a mathematical model for simulating the 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novel coronavirus (covid- ) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis key: cord- - l tatwq authors: malinovskaya, anna; otto, philipp title: online network monitoring date: - - journal: nan doi: nan sha: doc_id: cord_uid: l tatwq the application of network analysis has found great success in a wide variety of disciplines; however, the popularity of these approaches has revealed the difficulty in handling networks whose complexity scales rapidly. one of the main interests in network analysis is the online detection of anomalous behaviour. to overcome the curse of dimensionality, we introduce a network surveillance method bringing together network modelling and statistical process control. our approach is to apply multivariate control charts based on exponential smoothing and cumulative sums in order to monitor networks determined by temporal exponential random graph models (tergm). this allows us to account for potential temporal dependence, while simultaneously reducing the number of parameters to be monitored. the performance of the proposed charts is evaluated by calculating the average run length for both simulated and real data. to prove the appropriateness of the tergm to describe network data some measures of goodness of fit are inspected. we demonstrate the effectiveness of the proposed approach by an empirical application, monitoring daily flights in the united states to detect anomalous patterns. the digital information revolution offers a rich opportunity for scientific progress; however, the amount and variety of data available requires new analysis techniques for data mining, interpretation and application of results to deal with the growing complexity. as a consequence, these requirements have influenced the development of networks, bringing their analysis beyond the traditional sociological scope into many other disciplines, as varied as are physics, biology and statistics (cf. amaral et al. ; simpson et al. ; chen et al. ). one of the main interests in network study is the detection of anomalous behaviour. there are two types of network monitoring, differing in the treatment of nodes and links: fixed and random network surveillance (cf. leitch et al. ) . we concentrate on the modelling and monitoring of networks with randomly generated edges across time, describing a surveillance method of the second type. when talking about anomalies in temporal networks, the major interest is to find the point of time when a significant change happened and, if appropriate, to identify the vertices, edges or graph subsets which considerably contributed to the change (cf. akoglu et al. ). further differentiating depends on at least two factors: characteristics of the network data and available time granularity. hence, given a particular network to monitor it is worth first defining what is classified as "anomalous". to analyse the network data effectively and plausibly, it is important to account for its complex structure and the possibly high computational costs. our approach to mitigate these issues and simultaneously reflect the stochastic and dynamic nature of networks is to model them applying a temporal random graph model. we consider a general class of exponential random graph models (ergm) (cf. frank and strauss ; robins et al. ; schweinberger et al. ) , which was originally designed for modelling cross-sectional networks. this class includes many prominent random network configurations such as dyadic independence models and markov random graphs, enabling the ergm to be generally applicable to many types of complex networks. hanneke et al. ( ) developed a powerful dynamic extension based on ergm, namely the temporal exponential random graph model (tergm). these models contain the overall functionality of the ergm, additionally enabling time-dependent covariates. thus, our monitoring procedure for this class of models allows for many applications in different disciplines which are interested in analysing networks of medium size, such as sociology, political science, engineering, economics and psychology (cf. carrington et al. ; ward et al. ; das et al. ; jackson ; fonseca-pedrero ) . in the field of change detection, according to basseville et al. ( ) there are three classes of problems: online detection of a change, off-line hypotheses testing and off-line estimation of the change time. our method refers to the first class, meaning that the change point should be detected as soon as possible after it occurred. in this case, real-time monitoring of complex structures becomes necessary: for instance, if the network is observed every minute, the monitoring procedure should be faster than one minute. to perform online surveillance for real-time detection, the efficient way is to use tools from the field of statistical process control (spc). spc corresponds to an ensemble of analytical tools originally developed for industrial purposes, which is applied for achievement of process stability and variability reduction (e.g., montgomery ) . the leading spc tool for analysis is a control chart, which exists in various forms in terms of the number of variables, data type and different statistics being of interest. for example, the monitoring of network topology statistics applying the cumulative sum (cusum) chart and illustrating its effec- present a comparative study of univariate and multivariate ewma for social network monitoring. an overview of further studies is provided by noorossana et al. ( ) . in this paper, we present an online monitoring procedure based on the spc concept, which enables one to detect significant changes in the network structure in real time. the foundations of this approach together with the description of the selected network model and multivariate control charts are discussed in section . section outlines the simulation study and includes performance evaluation of the designed control charts. in section we monitor daily flights in the united states and explain the detected anomalies. we conclude with a discussion of outcomes and present several directions for future research. network monitoring is a form of an online surveillance procedure to detect deviations from a so-called in-control state, i.e., the state when no unaccountable variation of the process is present. this is done by sequential hypothesis testing over time, which has a strong connection to control charts. in other words, the purpose of control charting is to identify occurrences of unusual deviation of the observed process from a prespecified target (or in-control) process, distinguishing common from special causes of variation (cf. johnson and wichern ) . to be precise, the aim is to test the null hypothesis h ,t : the network observed at time point t is in its in-control state against the alternative h ,t : the network observed at time point t deviates from its in-control state. in this paper, we concentrate on monitoring of networks, which are modelled by the tergm that is briefly described below. the network (also interchangeably called "graph") is presented by its adjacency matrix y := (y i j ) i, j= ,...,n , where n represents the total number of nodes. two vertices (or nodes) i, j are adjacent if they are connected by an edge (also called a tie or link). in this case, y i j = , otherwise, y i j = . in case of an undirected network, y is symmetric. the connections of a node with itself are mostly not applicable to the majority of the networks, therefore, we assume that y ii = for all i = , . . . , n. formally, we define a network model as a collection {p θ (y ), y ∈ y : θ ∈ Θ}, where y denotes the ensemble of possible networks, p θ is a probability distribution on y and θ is a vector of parameters, ranging over possible values in the real-valued space Θ ⊆ ir p with p ∈ in (kolaczyk, ). this stochastic mechanism determines which of the n(n − ) edges (in case of directed labelled graphs) emerge, i.e., it assigns probabilities to each of the n(n− ) graphs (see cannings and penman, ) . the ergm functional representation is given by where y is the adjacency matrix of an observed graph with s : y → ir p being a p-dimensional statistic describing the essential properties of network based on y (cf. frank, ; wasserman and pattison, ) . there are several types of network terms, including dyadic dependent terms, for example, a statistic capturing transitivity, and dyadic independent terms, for instance, a term describing graph density (morris et al., ) . the parameters θ can be defined as respective coefficients of s(y ) which are of considerable interest in understanding the structural properties of a network. they reflect, on the network-level, the tendency of a graph to exhibit certain sub-structures relative to what would be expected from a model by chance, or, on the tie-level, the probability to observe a specific edge, given the rest of the graph (block et al., ) . the last interpretation follows from the representation of the problem as a log-odds ratio. the normalising constant in the denominator ensures that the sum of probabilities is equal to one, meaning it includes all possible network configurations in dynamic network modelling, a random sequence of y t for t = , , . . . with y t ∈ y defines a stochastic process for all t. it is possible that the dimensions of y t differ across the time stamps. to conduct surveillance over y t , we propose to consider only the dynamically estimated parameters of a random graph model in order to reduce computational complexity and to allow for real-time monitoring. in most of the cases, the dynamic network models serve as an extension of well-known static models. similarly, the discrete temporal expansion of the ergm is known as tergm (cf. hanneke et al., ) and can be seen as further advancement of a family of network models proposed by robins and pattison ( ) . the tergm defines the probability of a network at the discrete time point t both as a function of counted subgraphs in t and by including the network terms based on the previous graph observations until the particular time point t − v, that is where v represents the maximum temporal lag, capturing the networks which are incorporated into the θ estimation at t, hence, defining the complete temporal dependence of y t . we assume the markov structure between the observations, meaning (y t ⊥ ⊥ {y , . . . , y t− }|y t− ) (hanneke et al., ) . in this case, the network statistics s(·) include "memory terms" such as dyadic stability or reciprocity (leifeld et al., ) . the creation of a meaningful configuration of sufficient network statistics s(y ) replicates its ability to represent and reproduce the observed network close to the reality. its dimension can differ over the time, however, we assume that in each time stamp t we have the same network statistics s(·). in general, the selection of terms extensively depends on the field and context, although the statistical modelling standards such as avoidance of linear dependencies among the terms should be also considered (morris, handcock, and hunter, ). an improper selection can often lead to a degenerate model, i.e., when the algorithm does not converge consistently (cf. handcock, ; schweinberger, ) . in this case, as well as fine-tuning the configuration of statistics, one can modify some settings which design the estimation procedure of the model parameter, for example, the run time, the sample size or the step length (morris et al., ) . currently, there are two widely used approaches: chain monte carlo (mcmc) ml estimation (leifeld et al., ) . another possibility would be to add some robust statistics such as geometrically-weighted edgewise shared partnerships (gwesp) (snijders et al., ) . however, the tergm is less prone to the degeneracy issues as ascertained by leifeld and cranmer ( ) and hanneke et al. ( ) . regarding the selection of network terms, we assume that most of the network surveillance studies can reliably estimate beforehand the type of anomalies which are possible to occur. this assumption guides the choice of terms in the models throughout the paper. let p be the number of network statistics, which describe the in-control state and can reflect the deviations in the out-of-control state. thus, there are p variablesθ t = (θ t , . . . ,θ pt ) , namely the estimates of the network parameters θ at time point t. that is, we apply a moving window approach, where the coefficients are estimated at each time point t using the current and past z observed networks. moreover, let f θ ,Σ be the target distribution of these estimates with θ = e (θ , . . . ,θ p ) being the expected value and Σ the respective p × p variance-covariance matrix (montgomery, ) . we also assume that the temporal dependence is fully captured by the past z observed networks. thus, where τ denotes a change point to be detected and θ = θ . if τ = ∞ the network is set to be incontrol, whereas it is out of control in the case of τ ≤ t < ∞. furthermore, we assume that the estimation precision of the parameters does not change across t, i.e., Σ is constant for the in-control and out-of-control state. hence, the monitoring procedure is based on the expected values ofθ t . in fact, we can specify the above mentioned hypothesis as follows typically, a multivariate control chart consists of the control statistic depending on one or more characteristic quantities, plotted in time order, and a horizontal line, called the upper control limit (ucl) that indicates the amount of acceptable variation. a hypothesis h is rejected if the control statistic is equal to or exceeds the value of the ucl. hence, to perform monitoring a suitable control statistic and ucl are needed. subsequently, we discuss several control statistics and present a method to determine the respective ucls. the strength of the multivariate control chart over the univariate control chart is the ability to monitor several interrelated process variables. it implies that the corresponding test statistic should take into account the correlations of the data, be dimensionless and scale-invariant, as the process variables can considerably differ from each other. the squared mahalanobis distance, which represents the general form of the control statistic, fulfils these criteria and is defined as being the part of the respective "data depth" expression -mahalanobis depth that measures a deviation from an in-control distribution (cf. liu, ) . hence, d ( ) t maps the p-dimensional characteristic quantityθ t to an one-dimensional measure. it is important to note that the characteristic quantity at time point t is usually the mean of several samples at t, but in our case, we only observe one network at each instant of time. thus, the characteristic quantityθ t is the value of the obtained estimates and not the average of several samples. firstly, multivariate cusum (mcusum) charts (cf. woodall and ncube, ; joseph et al. ( ) ; ngai and zhang, ) may be used for network monitoring. one of the widely used version was proposed by crosier ( ) and is defined as follows where given that r = and k > . the respective chart statistic is and it signals if d ( ) t is greater than or equals the ucl. certainly, the values k and ucl considerably influence the performance of the chart. the parameter k, also known as reference value or allowance, reflects the variation tolerance, taking into consideration δ -the deviation from the mean measured in the standard deviation units we aim to detect. according to page ( ) and crosier ( ) , the chart is approximately optimal if k = δ / . secondly, we consider multivariate charts based on exponential smoothing (ewma). lowry et al. ( ) proposed a multivariate extension of the ewma control chart (mewma), which is defined as follows with the < λ ≤ and l = (cf. montgomery, ) . the corresponding chart statistic is where the covariance matrix is defined as together with the mcusum, the mewma is an advisable approach for detecting relatively small but persistent changes. however, the detection of large shifts is also possible by setting k or λ high. for instance, in case of the mewma with λ = , the chart statistic coincides with d ( ) t . thus, it is equivalent to the hotelling's t control procedure, which is suitable for detection of substantial deviations. it is worth to mention that the discussed methods are directionally invariant, therefore, the investigation of the data at the signal time point is necessary if the change direction is of particular interest. is equal to or exceeds the ucl, it means that the charts signal a change. to determine the ucls, one typically assumes that the chart has a predefined (low) probability of false alarms, i.e., signals when the process is in control, or a prescribed in-control average run length arl , i.e., the number of expected time steps until the first signal. to compute the ucls corresponding to arl , a prevalent number of multivariate control charts require a normally distributed target process (cf. johnson and wichern, ; porzio and ragozini, ; montgomery, ) . in our case, this assumption would need to be valid for the estimates of the network model parameters. however, while there are some studies on the distributions of particular network statistics (cf. yan and xu, ; yan et al., ; sambale and sinulis, ) , only a few results are obtained about the distribution of the parameter estimates. primarily, the difficulties to determine the distribution is that the assumption of i.i.d. (independent and identically distributed) data is violated in the ergm case. in addition, the parameters depend on the choice of the model terms and network size (he and zheng, ) . kolaczyk and krivitsky ( ) proved asymptotic normality for the ml estimates in a simplified context of the ergm, pointing out the necessity to establish a deeper understanding of the distributional properties of parameter estimates. thus, we do not rely on any distributional assumption, but determine the ucls via monte-carlo simulations in section . . to verify the applicability and effectiveness of the discussed approach, we design a simulation study followed by the surveillance of real-world data with the goal to obtain some insights into its temporal development. in practice, the in-control parameters θ and Σ are usually unknown and therefore have to be estimated. thus, one subdivides the sequence of networks into phase i and phase ii. in phase i, the process must coincide with the in-control state. thus, the true in-control parameters θ and Σ can be estimated by the sample mean vectorθ and the sample covariance matrix s of the estimated parametersθ t in phase i. using these estimates, the ucl is determined via simulations of the in-control networks, as we will show in the following part. it is important that the phase i replicates the natural behaviour of a network, so that if the network constantly grows, then it is vital to consider this aspect in phase i. similarly, if the type of network is prone to stay unchangeable in terms of additive connections or topological structure, this fact should be captured in phase i for reliable estimation and later network surveillance. after the necessary estimators of θ , Σ and the ucl are obtained, the calibrated control chart is applied to the actual data in phase ii. in specific cases of the constantly growing/topologically changing networks, we recommend to recalibrate the control chart after the length of arl to guarantee a trustworthy detection of the outliers. to be able to computeθ and s, we need a certain number of in-control networks. for this purpose, we generate temporal graph sequences of desired length t < τ, where each graph consists of n = nodes. the parameter τ defines the time stamp when an anomalous change is implemented. the simulation of synthetic networks is based on the markov chain principle: in the beginning, a network which is called the "base network" is simulated by applying an ergm with predefined network terms, so that it is possible to control the "network creation" indirectly. in our case, we select three network statistics, namely an edge term, a triangle term and a parameter that defines asymmetric dyads. subsequently, a fraction φ of elements of the adjacency matrix are randomly selected and set where m i j, denotes the probability of a transition from i to j in the in-control state. next, we need to guarantee that the generated samples of networks behave according to the requirements of phase i, i.e., capturing only the usual variation of the target process. for this purpose, we can exploit markov chain properties and calculate its steady state equilibrium vector π, as it follows that the expected number of edges and non-edges is given by π. using eigenvector decomposition, we find the steady state to be π = ( . , . ) . consequently, the expected number of edges in the graph in its steady state is . there are several possibilities to guarantee a generation of appropriate networks. in our case, we simulate networks in a burn-in period, such that the in-control state of phase i starts at t = . nevertheless, the network density is only one of the aspects to define the in-control process, as the temporal development and the topology are also involved in the network creation. each network in time point y t is simulated from the network y t− by repeating the steps described above. after the generation stage, the coefficients of the network statistics and of an additional term which describes the stability of both edges and non-edges over time with v = are estimated by applying a tergm with a certain window size z. the chosen estimation method is the bootstrap mple which is appropriate to handle a relatively large number of nodes and time points (leifeld et al., ) . eventually, we calibrate different control charts by computingθ, s, and the respective ucl via the bisection method. for two window sizes z = { , }, table in the next step, we analyse the performance of the proposed charts in terms of their detection speed. for this reason, we generate samples from phase ii, where t ≥ τ. the focus is on the detection of mean shifts, which are driven by an anomalous change in following three parameters: the vector of coefficients related to network termsθ t , the fraction of the randomly selected adjacency matrix entries φ and the transition matrix m . hence, we subdivide these scenarios into three different anomaly types which are briefly described in the chart flow presented in figure . we define a type anomaly as a persistent change in the values of m . that is, there is a transition matrix m = m when t ≥ τ. furthermore, we consider anomalies of type by introducing a new value φ in the generation process when t ≥ τ. anomalies of type differ from the previous two as it represents a "point change" -the abnormal behaviour occurs only at a single point of time but its outcome affects further development of the network. we recreate this type of anomalies by converting a fraction ζ of asymmetric edges into mutual links. this process happens at time point τ only. afterwards, the new networks are created similar to phase i by applying m and φ up until the anomaly is detected. all cases of different magnitude are summarised in table . as a performance measure we calculate the conditional expected delay (ced) of detection, conditional on a false signal not having been occurred before the ( should be detected (case . , . ). again, the reference/smoothing parameter should be chosen according to the expected shift size. for changes of the proportion of mutual edges, anomalies of type , the charts have different behaviour. first of all, the mewma chart outperforms in all cases except . and . with z = . however, the hotelling's chart functions clearly worse in the first two cases having a shorter window size. thus, we would recommend choosing λ = . if the change in the network topology is relatively small as in case . . in the opposite case of a larger change, λ could be chosen higher depending on the expected size of the shift, so that the control statistic also incorporates previous values. the disadvantage of both approaches is that small and persistent changes are not detected quickly when the parameters k or λ are not optimally chosen. for example, in figure , we can notice that the ced slightly exceeds the arl reflecting the poor performance. however, a careful selection of the parameters and the window size can overcome this problem. to summarise, the effectiveness of the presented charts to detect structural changes depends significantly on the accurate estimation of the anomaly size one aims to detect. thus, to ensure that no anomalies were missed, it can be effective to apply paired up charts and benefit from the strengths of each of them to detect varying types and sizes of anomalies, if the information on the possible change is not available or not reliable. to demonstrate applicability of the described method, the daily flight data of the united states through territories which allow travelling. that means, instead of having a direct journey from one geographical point to another, currently the route passes through several locations, which can be interpreted as nodes. thus, the topology of the graph has changed: instead of directed mutual links, the number of intransitive triads and asymmetric links start to increase significantly. we can incorporate both terms, together with the edge term and a memory term (v = ), and expect the estimates of the respective coefficients belonging to the first two statistics to be close to zero or strongly negative in the in-control case. initially, we need to decide which data are suitable to define observations coming from phase i, the estimates θ t of the tergm described by a series of boxplots in figure , we can observe extreme changes in the values. before proceeding with the analysis, it is important to evaluate whether a tergm fits the data well . for each of the years, we randomly selected one period of the length z and simulated networks based on the parameter estimates from each of the corresponding networks. to select appropriate control charts, we need to take into consideration specifications of the flight network data. firstly, it is common to have - travel peaks per year around holidays, which are not explicitly modelled, so that we can detect these changes as verifiable anomalous patterns. it is worth noting that one could account for such seasonality by including nodal or edge covariates. secondly, as we aim to detect considerable deviations from the in-control state, we are more interested the horizontal red line corresponds to the upper control limit and the red points to the occurred signals. in sequences of signals. thus, we have chosen k = . for mcusum and λ = . for the mewma chart. the target arl is set to days, therefore, we could expect roughly . in-control signals per year by construction of the charts. to identify smaller and more specific changes in the daily flight data of the us, one could also integrate nodal and edge covariates which would refer to further aspects of the network. alternatively, control charts with smaller k and λ can be applied. statistical methods can be remarkably powerful for the surveillance of networks. however, due to the complex structure and possibly large size of the adjacency matrix, traditional tools for multivariate process control cannot directly be applied, but the network's complexity must be reduced first. for instance, this can be done by statistical modelling of the network. the choice of the model is crucial as it decides constraints and simplifications of the network which later influence the types of changes we are able to detect. in this paper, we show how multivariate control charts can be used to detect changes in tergm networks. the proposed methods can be applied in real time. this general approach is applicable for various types of networks in terms of the edge direction and topology, as well as allows for the integration of nodal and edge covariates. additionally, we make no assumptions on distribution and account for temporal dependence. the performance of our procedure is evaluated for different anomalous scenarios by comparing the ced of the calibrated control charts. according to the classification and explanation of anomalies provided by ranshous et al. ( ) , the surveillance method presented in this paper is applicable for event and point change detection in temporal networks. the difference between these problems lies in the duration of the abnormal behaviour: while change points indicate a time point when the anomaly is persistent until the next change point, events indicate short-term incidents, after that the network returns to its natural state. eventually, we illustrated the applicability of our approach by monitoring daily flights in the united states. both control charts were able to detect the beginning of the lock-down period due to the covid- pandemic. the mewma chart signalled a change just two days after a level "no travel" warning was issued. despite the benefits of the tergm, such as incorporation of the temporal dimension and representation of the network in terms of its sufficient statistics, there are several considerable drawbacks. other than the difficulty to determine a suitable combination of the network terms, the model is not suitable for networks of large size (block et al., ) . furthermore, the temporal dependency statistics in the tergm depend on the selected temporal lag and the size of the time window over which the data is modelled (leifeld and cranmer, ) . thus, the accurate modelling of the network strongly relies on the analyst's knowledge about its nature. a helpful extension of the approach would be the implementation of the separable temporal exponential random graph model (stergm) that subdivides the network changes into two distinct streams (cf. krivitsky and handcock, ; fritz et al., ) . in this case, it could be possible to monitor the dissolution and formation of links separately, so that the interpretation of changes in the network would become clearer. regarding the multivariate control charts, there are also some aspects to consider. referring to montgomery ( ) , the multivariate control charts perform well if the number of process variables is not too large, usually up to . also, a possible extension of the procedure is to design a monitoring process when the values for Σ can vary between the in-control and out-of-control states. whether this factor would beneficially enrich the surveillance remains open for future research. in our case, we did not rely on any distributional assumptions of the parameters, but we used simulation methods to calibrate the charts. hence, further development of adaptive control charts with different characteristics is interesting as they could remarkably improve the performance of the anomaly detection (cf. sparks and wilson, ). graph based anomaly detection and description: a survey classes of small-world networks detection of abrupt changes: theory and application change we can believe in: comparing longitudinal network models on consistency, interpretability and predictive power models of random graphs and their applications models and methods in social network analysis tail event driven networks of sifis multivariate 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detecting a change statistical analysis of network data on the question of effective sample size in network modeling: an asymptotic inquiry. statistical science: a a separable model for dynamic networks a theoretical and empirical comparison of the temporal exponential random graph model and the stochastic actor-oriented model temporal exponential random graph models with btergm: estimation and bootstrap confidence intervals toward epidemic thresholds on temporal networks: a review and open questions control charts for multivariate processes analyzing dynamic change in social network based on distribution-free multivariate process control method a multivariate exponentially weighted moving average control chart detecting change in longitudinal social networks statistical quality control specification of exponential-family random graph models: terms and computational aspects multivariate cumulative sum control charts based on projection pursuit an overview of dynamic anomaly detection in social networks via control charts continuous inspection schemes multivariate control charts from a data mining perspective. recent advances in data mining of enterprise data anomaly detection in dynamic networks: a survey random graph models for temporal processes in social networks an introduction to exponential random graph (p*) models for social networks monitoring of social network and change detection by applying statistical process: ergm change point detection in social networks using a multivariate exponentially weighted moving average chart logarithmic sobolev inequalities for finite spin systems and applications instability, sensitivity, and degeneracy of discrete exponential families exponential-family models of random graphs: inference in finite-, super-, and infinite population scenarios analyzing complex functional brain networks: fusing statistics and network science to understand the brain new specifications for exponential random graph models monitoring communication outbreaks among an unknown team of actors in dynamic networks network analysis and political science logit models and logistic regressions for social networks: i. an introduction to markov graphs and p* modeling and detecting change in temporal networks via the degree corrected stochastic block model multivariate cusum quality-control procedures a central limit theorem in the β -model for undirected random graphs with a diverging number of vertices asymptotics in directed exponential random graph models with an increasing bi-degree sequence key: cord- - mfuwx l authors: seelig, frederik; bezerra, haroldo; cameron, mary; hii, jeffrey; hiscox, alexandra; irish, seth; jones, robert t.; lang, trudie; lindsay, steven w.; lowe, rachel; nyoni, tanaka manikidza; power, grace m.; quintero, juliana; stewart-ibarra, anna m.; tusting, lucy s.; tytheridge, scott; logan, james g. title: the covid- pandemic should not derail global vector control efforts date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: mfuwx l nan the covid- pandemic is placing immense pressure on health systems worldwide. this is particularly apparent in resource-poor settings with limited capacity to treat and contain new disease outbreaks. the world health organization (who) has emphasised the crucial need to sustain efforts to prevent, detect, and treat malaria during this pandemic [ ] . however, a similar approach should also be adopted for the control of arboviral diseases of global importance, including dengue, zika, chikungunya, and yellow fever, as recommended by the pan-american health organization (paho) in their interim guidance on control of aedes aegypti mosquitos during the covid- pandemic [ ] . for example, an unprecedented dengue epidemic continues to affect latin america and the caribbean, with a surge of cases experienced in the first half of [ ] . even before the covid- pandemic began, many countries were struggling to control dengue. the public health interventions initiated to halt the covid- spread have already severely affected routine vector surveillance and control activities (such as regular household surveys) [ , ] . the combined impact of both covid- and epidemics of dengue or other vector-borne diseases (vbds) could have potentially devastating consequences [ ] . in view of these combined challenges, we reiterate our solidarity with the global partners who are dealing with the covid- pandemic, while strongly urging them to consider these recommendations for the control of vbds: • continue the implementation of the who's global vector control response - (gvcr) strategy and regional policies for vector control [ , ] , with respect to inter-and intrasectoral collaboration, engagement and mobilisation of communities, and scaling up of vector control if required, according to the implementation plan of vector control activities, while adapting activities as necessary to prevent further spread of covid- , in particular vector surveillance, which may need to be scaled down [ , ] . and, more specifically with respect to vbd control, we present a list of recommended actions grouped into three categories: . maintain vector control operations in the context of covid- (adapting activities to improve worker safety and to prevent further spread): a. continue and enhance protection of vulnerable populations against vbds, e.g., through providing topical repellents and distributing bed nets, and deliver vector control safely while practising physical distancing. b. ensure the safety of community workers and volunteers through training on safe methods to conduct their work in the face of covid- and provide personal protective equipment (see section . in [ ] for further details). c. perform intensive risk assessment and contingency planning for continuity of vector control by dedicated health personnel with appropriate training. this may involve moving from community-based control to household-, clinic-, hospital-, or school-level control measures. a. encourage global funding bodies and dedicated donors to provide long-term funding for capacity building in vector surveillance and control, including training and retention of local staff, and for contingencies during any anticipated lockdown. it is imperative not to reduce existing funding on vector control programmes or to lose sight of the value of vector control, which would offset any previous gains. b. improve the validity, availability, and proper use of rapid testing, both for covid- and vbds, to prevent misdiagnosis and to improve case management for potential coinfections and comorbidities [ ] . c. identify gaps in practices and knowledge related to vector-borne disease control and encourage local and national stakeholders to address these deficiencies. d. improve preparedness for outbreaks (vector-borne or not) by devising a comprehensive preparedness plan that will identify all activities and materials required for each stage (from no risk to epidemic) and by providing strategic reserves of pharmaceuticals, personal protective equipment, public health insecticides, and vector control equipment. e. despite significant progress to date, many research activities are currently on hold. the search for new and effective tools for both malaria and dengue control must continue, including the use of artificial intelligence and novel technological solutions, such as drones, to assist in vector surveillance and control. f. integrate modelling to predict the combined impact of vbds and covid- . . covid- disease monitoring and control innovations that could be applied to vector control: a. one of the encouraging aspects of the response to the covid- pandemic has been the rapid and open global dissemination of data, such as virus sequence data. this has allowed development of the first vaccine candidates in record time. we strongly recommend a similar effort to promote relevant data sharing on vectors, arboviruses, and other vbds to support public health interventions. the global vector hub (gvh) online platform was designed to facilitate easy access and exchange of information, data, and resources among relevant stakeholders, and an early version was launched in june to support global vector control efforts in the context of the covid- pandemic. b. provide accurate and up-to-date information and data from trusted sources to fight the spread of misinformation. c. support studies that map and evaluate the impact of distancing and isolation measures on vector control interventions and vbds, especially in large urban centres. it is expected that the most vulnerable and poorest populations will suffer most from covid- , due to lack of access to appropriate care and the impacts of isolation measures on fragile livelihoods. it is vital that the covid- response does not increase vbd threats in these communities by derailing global vector control efforts. who urges countries to ensure the continuity of malaria services in the context of the covid- pandemic control of aedes aegypti in the scenario of simultaneous transmission of covid- pan american health organization/world health organization covid- in latin america: the implications of the first confirmed case in brazil dengue kills too'-latin america faces two epidemics at once. reuters world news preventing dengue epidemics during the covid- pandemic action plan on entomology and vector control - . th directing council of paho, th session of the who regional committee for the americas. washington, d.c.: paho, (resolution cd . ) world health organization. tailoring malaria interventions in the covid- response. geneva: who, community-based health care, including outreach and campaigns, in the context of the covid- pandemic. geneva: who, covert covid- and false-positive dengue serology in singapore the opinions expressed in this letter are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention and pan-american health organization/world health organization. key: cord- -z kvf n authors: rogerson, stephen j.; beeson, james g.; laman, moses; poespoprodjo, jeanne rini; william, timothy; simpson, julie a.; price, ric n. title: identifying and combating the impacts of covid- on malaria date: - - journal: bmc med doi: . /s - - -x sha: doc_id: cord_uid: z kvf n background: the covid- pandemic has resulted in millions of infections, hundreds of thousands of deaths and major societal disruption due to lockdowns and other restrictions introduced to limit disease spread. relatively little attention has been paid to understanding how the pandemic has affected treatment, prevention and control of malaria, which is a major cause of death and disease and predominantly affects people in less well-resourced settings. main body: recent successes in malaria control and elimination have reduced the global malaria burden, but these gains are fragile and progress has stalled in the past years. withdrawing successful interventions often results in rapid malaria resurgence, primarily threatening vulnerable young children and pregnant women. malaria programmes are being affected in many ways by covid- . for prevention of malaria, insecticide-treated nets need regular renewal, but distribution campaigns have been delayed or cancelled. for detection and treatment of malaria, individuals may stop attending health facilities, out of fear of exposure to covid- , or because they cannot afford transport, and health care workers require additional resources to protect themselves from covid- . supplies of diagnostics and drugs are being interrupted, which is compounded by production of substandard and falsified medicines and diagnostics. these disruptions are predicted to double the number of young african children dying of malaria in the coming year and may impact efforts to control the spread of drug resistance. using examples from successful malaria control and elimination campaigns, we propose strategies to re-establish malaria control activities and maintain elimination efforts in the context of the covid- pandemic, which is likely to be a long-term challenge. all sectors of society, including governments, donors, private sector and civil society organisations, have crucial roles to play to prevent malaria resurgence. sparse resources must be allocated efficiently to ensure integrated health care systems that can sustain control activities against covid- as well as malaria and other priority infectious diseases. conclusion: as we deal with the covid- pandemic, it is crucial that other major killers such as malaria are not ignored. history tells us that if we do, the consequences will be dire, particularly in vulnerable populations. the impact of the covid- pandemic on the control of infectious diseases is substantial, undermining established programmes addressing hiv, tuberculosis and malaria and childhood vaccination. this opinion piece focuses on the threat covid- poses to the control of malaria and the steps that can be taken to mitigate these impacts. over the past years, major gains have been made in reducing the global burden of malaria, with countries achieving malaria elimination. these gains are largely attributable to expanding the distribution of insecticidetreated bed nets (itns), indoor spraying of residual insecticides (irs) and other vector control strategies; access to early diagnosis (e.g. rapid diagnostic tests (rdts)); and more effective antimalarial treatments [ ] , together with targeted interventions such as intermittent preventive treatment in pregnancy (iptp) and seasonal malaria chemoprevention (smc). this multipronged approach has been enabled by a greater political, financial and global commitment to malaria elimination, encouraged by ambitious targets, such as reducing malaria globally by > % by (compared to ), eliminating malaria from the asia pacific by and africa being largely malaria-free by . however, in recent years, progress in reducing the global burden of malaria has stalled. in , there were an estimated million cases, compared with million in , and over , deaths [ ] . challenges in achieving malaria elimination include the emergence and spread of drug-resistant parasites and insecticideresistant mosquitos, suboptimal rdts, lack of universal access to malaria prevention and treatment and the lack of a highly effective vaccine. malaria funding is below what is required to achieve global goals, and many countries face competing health priorities in the context of severely constrained resources [ ] ; tuberculosis, human immunodeficiency virus (hiv) infection and other diseases face similar challenges. in this environment, the emergence and spread of covid- presents a huge threat to malaria control that could reverse recent gains in many malaria-endemic countries. historically, curtailing malaria control activities has been followed by resurgence in malaria morbidity and mortality [ ] . this has occurred when programmes were reduced due to funding constraints or disrupted by war, disaster or conflict [ ] . following the termination of a dichlorodiphenyltrichloroethane (ddt) programme in indonesia in the s, annual malaria cases rose from < to , . between and , sri lanka reduced malaria cases from . million to just , but experienced a massive resurgence to over , cases per year [ ] . while sri lanka has now successfully eliminated malaria, other asian countries that are approaching elimination face similar risks of resurgence if current programmes are substantially disrupted by the covid- pandemic. economic collapse and health system failure are known to be critical causes of rising morbidity and mortality from infectious diseases, and this commonly spreads beyond national borders. in venezuela, the recent economic crisis has been accompanied by population movements and major increases in vector-borne diseases, including a fivefold increase in malaria cases [ ] ; venezuela now has over half the malaria cases in the americas [ ] , and in adjacent regions of colombia and brazil, up to % of malaria cases are attributed to recent migration [ ] . covid- has already caused major disruption to economic activity, which could contribute to malaria resurgence. during the ebola fever crisis in west africa, excess malaria deaths outnumbered total deaths from ebola [ ] . deaths from hiv infection and tuberculosis also surged [ ] . contributing factors, including deaths of health care workers, overwhelmed health facilities and fear of contracting disease at health services [ , ] , are relevant to covid- . the ebola epidemic disrupted distribution of itns and resulted in increased malaria transmission, while poor access to malaria treatment led to dramatic increases in deaths in children [ ] . subsequent modelling studies indicated that additional itn distribution and introduction of safe monthly mass drug administration (mda) [ ] with dihydroartemisinin-piperaquine each had the potential to reduce malaria deaths during the epidemic by approximately two thirds [ ] . although international donors pledged support for ebola, significant delays occurred in getting funds on the ground and this contributed to the severity of the epidemic [ ] . likewise, the magnitude and timing of additional support to covid- -affected countries is critical. support for the management of covid- must be combined with support for malaria treatment and prevention programmes; it is likely that this will need to be sustained for years until covid- is brought under control. key interventions and innovative approaches, such as targeted mda programmes and enhanced distribution of itns, will be critical in preventing dramatic increases in malaria deaths [ ] , but their implementation and prioritisation will bring logistic and financial challenges given covid- disruptions and the competing needs of other health issues and services. while covid- is less often severe in children and pregnant women [ , ] , these groups would bear a disproportionate burden of excess malaria mortality arising from covid- -related disruption of health systems and malaria control programmes, particularly in sub-saharan africa. recently, the malaria atlas project modelled these potential impacts in africa for the world health organization's (who) global malaria program [ ] . a range of scenarios were considered, such as ceasing itn distribution campaigns planned for , reductions of routine itn distribution and reduced access to effective antimalarial drugs. in the worst-case scenario, a % decrease in itn distribution coupled with a % decrease in access to artemisinin combination therapies (acts) was predicted to result in a % increase in malaria cases, and doubling of malaria deaths within a year to , [ ] , % of them in children under . these models do not include additional increases in malaria that could result from disruptions in distribution of smc (which currently protects million children in countries) and iptp (which protects pregnant women and their babies in african countries from malaria in pregnancy and low birth weight) [ ] . neither do they include the potential impacts of decreasing irs and other vector control strategies, or the consequences of reassigning malaria personnel to covid- -related activities. the future severity of the impact of covid- in africa is unknown. at the time of writing, africa has % of the world's covid- cases, and % of its deaths [ ] , with around , infections and , deaths reported [ ] . these numbers are likely to be underestimates due to limited testing being undertaken in some regions. these rising numbers are occurring despite many countries taking early, decisive steps to lockdown borders and implement social distancing in crowded environments and other preventive measures. if these measures, or the disease itself, substantially impede normal functions of the health system, this could result in delayed treatment for young children, in whom severe malaria develops rapidly, even with prompt treatment - % of children with cerebral malaria die. if treatment is not available, staggering numbers of young children may lose their lives from malaria. covid- -related lockdowns threaten the livelihoods of the many africans who work in the informal sector, affecting their ability to pay for transport and for health care services when these are not free [ , ] . preventive and therapeutic maternal child health services such as antenatal clinics and childhood vaccination programmes (with > million infants at risk) are at risk, and economic disruptions may exacerbate child undernutrition [ ] . continued provision of chronic medications for tuberculosis and hiv (there are > million people living with hiv and aids in africa) and access to malaria treatment and prevention services (such as itn distribution, iptp and smc) collectively threaten major increases in infectious disease morbidity and mortality. while the greatest burden of malaria is in sub-saharan africa, two billion people in the asia pacific region remain at risk of malaria [ ] . this region has been the global hotspot for emergence of plasmodium falciparum resistant to drugs, including chloroquine, antifolates and mefloquine. the spread of chloroquine and antifolate resistance to africa reversed gains of intense malaria control activities undertaken between and , leading to increasing mortality and morbidity [ ] . in the s, the prospect of untreatable malaria was averted by the introduction of acts, which have now been adopted as first-line treatment by almost all malariaendemic countries [ ] . over the last decade, artemisininresistant p. falciparum, originating from western cambodia, has spread across the greater mekong subregion (gms). the efficacy of a key partner drug, piperaquine, has also declined dramatically [ , ] , causing the efficacy of dihydroartemisinin-piperaquine to fall below % in cambodia, thailand and vietnam [ ] , once again raising concerns of resurgent and untreatable malaria. while new antimalarial agents are urgently required, these are unlikely to be available for several years [ ] . who has a detailed strategy to contain artemisinin resistance, and substantial resources have been made available to eliminate drugresistant malaria before it spreads beyond the gms. from to , across who's south east asian region, there was a % reduction in malaria morbidity and a % reduction in mortality. these gains remain fragile and depend on robust health systems achieving early diagnosis and treatment of symptomatic patients and clearing parasite reservoirs from asymptomatic populations. if efforts to eliminate drug-resistant p. falciparum from the gms falter, resurgence is highly likely to be followed by the spread of resistance into south asia, increasing the risks that resistant malaria will spread to africa. in many places, community-based malaria workers work in close proximity to febrile patients and are at high risk of covid- . despite the risks, adequate personal protective equipment is often lacking, and workers suffer the stigma of being potential sources of viral infection. funds and personnel are being reassigned from malaria and other programmes to enable covid- response efforts. malaria elimination campaigns must reach marginalised groups living in remote and border areas [ ] , but these programmes are at particular risk of being scaled back for logistic or economic reasons associated with covid- , putting communities at risk. together, these complex issues are compromising the provision of health care and surveillance for malaria and threatening elimination efforts. to reduce the impact of covid- disruptions, it is essential that the supply of diagnostics and treatments for malaria are maintained and that there is strong support of itn distribution, irs and other preventive interventions. maintaining drug quality is also critical, with potential proliferation of substandard and falsified medicines and diagnostics when supply chains for established suppliers are disrupted [ ] . furthermore, companies may switch from producing drugs or diagnostics for malaria to covid- , driven by higher profit margins [ ] with clear negative consequences for malaria treatment and control. community education and engagement will be important to reinforce messages regarding malaria prevention, diagnosis and treatment. provision of health services that can provide prompt diagnosis and treatment of malaria is critical, together with professional support of health workers to ensure safe working environments and properly resourced facilities. ideally, these activities would be integrated within the covid- response [ ] . where adequate personal protective equipment or rdts are not available, presumptive malaria treatment may be required (based on symptoms of fever without another obvious cause) [ ] . this brings risks of confounding malaria and covid- and of missing other key diseases like childhood pneumonia. if health systems are not able to maintain malaria control interventions while managing the response to the covid- emergency, they are highly likely to be further impacted by additional malaria cases. a series of case studies prepared for the who in by the university of california san francisco [ ] documented effective strategies used to re-establish malaria control and support progress towards elimination in countries. political will is a critical component, with programmes supported by national governments, although implementation may be also be coordinated at local levels. while some countries are able to self-fund flexible programmes, many rely on donor support, particularly from the global fund for aids, tuberculosis and malaria, to assist with procuring diagnostics, drugs and itns. distribution channels must be strengthened, and capacity building is required to ensure that a skilled and adaptable workforce can deliver interventions. integrated approaches to control vector-borne diseases are essential, with cadres of health care workers benefiting from donor-supported malaria-specific training and other professional development [ ] . with regard to service delivery, strengthening the treatment and prevention of malaria must go hand-in-hand with overall health systems strengthening, as part of integrated models of primary health care. in areas of heterogeneous transmission, robust surveillance and targeted control activities can be highly effective. in countries approaching elimination, such responses will need to include localised vector control responses with focal irs campaigns and potentially other measures such as environmental management [ ] . intensified passive case detection and prompt disease notification can in turn facilitate targeted screening and active case detection programmes. a combination of reactive focal mass drug administration with artemether lumefantrine and reactive irs with primiphosmethyl was recently trialled in a low transmission area of namibia; each intervention halved clinical malaria cases, and the combination reduced malaria by % [ ] . management of malaria in border areas requires special initiatives. in some island nations, all arrivals from endemic countries are recorded and monitored, while countries in the end stages of elimination, such as bhutan, turkey and turkmenistan, have developed systems to track and identify infections in mobile populations including cross border traders, migrant workers and refugees. transnational cooperation around borders is key to tackling malaria in undocumented migrants, seasonal workers and marginalised populations in remote border areas. economic disruption following the covid- pandemic is likely to lead to increases in malaria cases or changing patterns of population movements in these groups. in high transmission areas, continuing malaria surveillance combined with real-time reporting will allow the prompt detection of hotspots of transmission. itn distributions and irs campaigns combined with mda hold great potential for bringing malaria under control. technical advances such as geographical information system (gis)-based case mapping and electronic reporting of cases can provide real-time data on numbers and locations of cases and tailored responses to high burden areas. in the context of covid- , surveillance may be difficult due to multiple factors, including restrictions on movements, concerns about field worker exposure, requirements for additional ppe or resource constraints. therefore, integrated, population-wide approaches that include itn distribution and mda and/or irs for malaria with treatment of neglected tropical diseases, and community health education, may be more practicable to maximise population protection. many successful malaria elimination campaigns have had a strong focus on community awareness and community participation. the latter may extend to active involvement in environmental management and breeding site interventions (as in réunion), and there is great potential to integrate community education and health promotion on malaria with covid- activities. recent years have seen the emergence of civil society organisations such as the civil society for malaria elimination [ ] and campaigns such as #zeromalariastartswithme, which are empowering community and civil society to work for effective, sustainable and inclusive malaria control solutions. close engagement with local community is integral to the implementation of high-impact programmes. private sector involvement is important on several levels. private clinics, pharmacies and local drug sellers have crucial roles in ensuring accurate diagnosis and prompt effective treatment of malaria. in many settings, these services also have important roles in reporting malaria infection [ ] . large enterprises in extractive and agricultural industries may house great numbers of workers and families, sometimes on a seasonal basis. these can be sources of local disease outbreaks or can help to strengthen the surrounding health care system and work jointly on malaria control activities. irrigation schemes require ongoing maintenance and oversight to prevent the development of vector breeding sites. partnerships between successful local companies and local health authorities can help to support specific malaria control activities of mutual interest, to "give back" to their home community. following the first covid- epidemic peak in malariaaffected countries, reinvigoration of malaria control will be needed in many regions (table ) , and steps will need to be taken to prepare for further waves of infection. strengthening surveillance for malaria is crucial together with campaigns to ensure sustainable provision of antimalarials and itns, while strategies are explored to allow community-based malaria programmes to continue. these efforts will inform health system responses. lessons can be learnt from childhood immunisation programmes, which have achieved significant successes in increasing immunisation coverage following disruptions through programme intensifications, outreach activities, community engagement and political support and leadership. transnational and regional initiatives, including operational research, will help to identify strategies for subsequent malaria control activities. as we struggle with covid- , we call on the leaders of countries across the world to recommit to malaria elimination as an achievable and enduring public good. the broader goal of malaria eradication remains highly attractive. an estimated us$ -$ billion investment would unlock over us$ trillion in economic benefits [ ] . if we reduce our focus on malaria, it will resurge, bringing a terrible death toll and even greater economic hardship. if efforts to eliminate drug-resistant p. falciparum malaria from the gms falter, resistant malaria will likely spread to africa, an outcome that could lead to a dramatic increase in childhood deaths [ ] . the world's most affluent countries are already grappling with the challenges of delivering health care and responding to the secondary social and economic costs of the covid- pandemic. in malaria-endemic countries, it is vital that measures are taken both to protect health workers and maintain malaria control activities. the global community cannot afford to cut aid to established programmes that have contributed significantly to the major progress made against malaria and other diseases; we must continue to look outwards, rather than turning inwards at the expense of the world's most vulnerable. successful malaria programmes that are depleted by the covid- epidemic must be rebuilt as quickly as possible to prevent a novel pathogen from giving a new lease of life to an old one. table priorities for combating malaria ensure the safety of health care workers and the populations they serve through adequate provision of personal protective equipment, hand hygiene and ability to practice social and physical distancing. provide resources to enable national malaria control programmes to continue to carry out established programmes. maintain campaigns and systems to procure and distribute itns, ensuring continuing coverage of high-risk populations. secure ongoing production and supply chains of quality-approved malaria diagnostics, treatments and preventives. ensure the timely delivery of these essential supplies to all health facilities. consider safely implementing campaigns of mass drug administration, especially during periods of peak malaria risk. support malaria-endemic countries both in fighting covid- disease and in controlling malaria through an integrated health care programme and community engagement. resume, and maintain funding for, non-covid- research, from discovery through to clinical, epidemiological and health system studies [ ] . maintain existing global funding for malaria control and elimination. recommit to the elimination of malaria from the asia pacific and renew efforts to achieve a malaria-free world. melbourne australia; and prof peter gething the effect of malaria control on plasmodium falciparum in africa between geneva: world health organization malaria elimination and eradication malaria epidemics: detection and control, forecasting and prevention. geneva: world health organization malaria resurgence: a systematic review and assessment of its causes venezuela's humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region malaria morbidity and mortality in ebola-affected countries caused by decreased health-care capacity, and the potential effect of mitigation strategies: a modelling analysis effects of response to - ebola outbreak on deaths from malaria, hiv/aids, and tuberculosis, west africa ebola: the hidden toll world health organization. guidance on temporary malaria control measures in ebola-affected countries international donations to the ebola virus outbreak: too little, too late? world health organization. tailoring malaria interventions in the covid- response. geneva: world health organization intensive care unit admissions for pregnant and non-pregnant women with covid- clinical and epidemiological features of children china: an observational cohort study world health organization. the potential impact of health service disruptions on the burden of malaria: a modelling analysis for countries in sub-saharan africa. geneva: world health organization covid- in africa: no room for complacency african centres for disease control and prevention what does the covid- pandemic mean for hiv, tuberculosis, and malaria control early estimates of the indirect effects of the covid- pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study measurement of trends in childhood malaria mortality in africa: an assessment of progress toward targets based on verbal autopsy world health organization. guidelines for the treatment of malaria. geneva: world health organization spread of artemisinin resistance in plasmodium falciparum malaria evolution and expansion of multidrug-resistant malaria in southeast asia: a genomic epidemiology study determinants of dihydroartemisinin-piperaquine treatment failure in plasmodium falciparum malaria in cambodia, thailand, and vietnam: a prospective clinical, pharmacological, and genetic study the antimalarial pipeline malaria elimination in remote communities requires integration of malaria control activities into general health care: an observational study and interrupted time series analysis in myanmar signatories from c. covid- and risks to the supply and quality of tests, drugs, and vaccines demand for coronavirus tests raises concerns over hiv and malaria the guardian novel coronavirus disease (covid- ) mitigation steps provide a blueprint for malaria control and elimination effectiveness of reactive focal mass drug administration and reactive focal vector control to reduce malaria transmission in the low malaria-endemic setting of namibia: a cluster-randomised controlled, open-label, two-by-two factorial design trial civil society for malaria elimination nonessential research in the new normal: the impact of novel coronavirus disease (covid- ) from aspiration to action: what will it take to end malaria? springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the acreme investigators who contributed to this article include prof nicholas anstey, menzies school authors' contributions sjr and jgb conceived the article. sjr, jgb, jas and rnp drafted the article with input from tw, jrp and ml. all authors read and approved the final version. acreme investigators provided comments and contributed to discussion. key: cord- -pz lsaw authors: rodrigues, helena sofia title: optimal control and numerical optimization applied to epidemiological models date: - - journal: nan doi: nan sha: doc_id: cord_uid: pz lsaw the relationship between epidemiology, mathematical modeling and computational tools allows to build and test theories on the development and battling of a disease. this phd thesis is motivated by the study of epidemiological models applied to infectious diseases in an optimal control perspective, giving particular relevance to dengue. dengue is a subtropical and tropical disease transmitted by mosquitoes, that affects about million people per year and is considered by the world health organization a major concern for public health. the mathematical models developed and tested in this work, are based on ordinary differential equations that describe the dynamics underlying the disease, including the interaction between humans and mosquitoes. an analytical study is made related to equilibrium points, their stability and basic reproduction number. the spreading of dengue can be attenuated through measures to control the transmission vector, such as the use of specific insecticides and educational campaigns. since the development of a potential vaccine has been a recent global bet, models based on the simulation of a hypothetical vaccination process in a population are proposed. based on optimal control theory, we have analyzed the optimal strategies for using these controls, and respective impact on the reduction/eradication of the disease during an outbreak in the population, considering a bioeconomic approach. the formulated problems are numerically solved using direct and indirect methods. the first discretize the problem turning it into a nonlinear optimization problem. indirect methods use the pontryagin maximum principle as a necessary condition to find the optimal curve for the respective control. in these two strategies several numerical software packages are used. a relação entre a epidemiologia, a modelação matemática e as ferramentas computacionais permite construir e testar teorias sobre o desenvolvimento e combate de uma doença. esta tese tem como motivação o estudo de modelos epidemiológicos aplicados a doenças infeciosas numa perspetiva de controloÓtimo, dando particular relevância ao dengue. sendo uma doença tropical e subtropical transmitida por mosquitos, afecta cerca de milhões de pessoas por ano, eé considerada pela organização mundial de saúde como uma grande preocupação para a saúde pública. os modelos matemáticos desenvolvidos e testados neste trabalho, baseiamse em equações diferenciais ordinárias que descrevem a dinâmica subjacentè a doença nomeadamente a interação entre humanos e mosquitos.É feito um estudo analítico dos mesmos relativamente aos pontos de equilíbrio, sua estabilidade e número básico de reprodução. a propagação do dengue pode ser atenuada através de medidas de controlo do vetor transmissor, tais como o uso de inseticidas específicos e campanhas educacionais. como o desenvolvimento de uma potencial vacina tem sido uma aposta mundial recente, são propostos modelos baseados na simulação de um hipotético processo de vacinação numa população. tendo por base a teoria de controloÓtimo, são analisadas as estratégiasótimas para o uso destes controlos e respetivas repercussões na redução/erradicação da doença aquando de um surto na população, considerando uma abordagem bioeconómica. os problemas formulados são resolvidos numericamente usando métodos diretos e indiretos. os primeiros discretizam o problema reformulando-o num problema de optimização não linear. os métodos indiretos usam o princípio do máximo de pontryagin como condição necessária para encontrar a curvá otima para o respetivo controlo. nestas duas estratégias utilizam-se vários pacotes de software numérico. ao longo deste trabalho, houve sempre um compromisso entre o realismo dos modelos epidemiológicos e a sua tratabilidade em termos matemáticos. introduction "mathematical biology is a fast-growing, well-recognized, albeit not clearly defined, subject and is, to my mind, the most exciting modern application of mathematics." -j. d. murray, mathematical biology, epidemiology has become an important issue for modern society. the relationship between mathematics and epidemiology has been increasing. for the mathematician, epidemiology provides new and exciting branches, while for the epidemiologist, mathematical modeling offers an important research tool in the study of the evolution of diseases. in , a smallpox model was proposed by daniel bernoulli and is considered by many authors the first epidemiological mathematical model. theoretical papers by kermack and mckendrinck, between and about infectious disease models, have had a great influence in the development of mathematical epidemiology models [ ] . most of the basic theory had been developed during that time, but the theoretical progress has been steady since then [ ] . mathematical models are being increasingly used to elucidate the transmission of several diseases. these models, usually based on compartment models, may be rather simple, but studying them is crucial in gaining important knowledge of the underlying aspects of the infectious diseases spread out [ ] , and to evaluate the potential impact of control programs in reducing morbidity and mortality. after the second world war, the strategy of public health has been focusing on the control and elimination of the organisms that cause the diseases. the appearance of new antibiotics and vaccines brought a positive perspective of the diseases eradication. however, factors such as resistance to the medicine by the microorganisms, demographic evolution, accelerated urbanization, increased travelling and climate change, led to new diseases and the resurgence of old ones. in , the human immunodeficiency virus (hiv) appears and since then, become as important sexually transmitted disease throughout the world [ ] . futhermore, malaria, tuberculosis, dengue and yellow fever have re-emerged and, as a result of climate changes, has been spreading into new regions [ ] . recent years have seen an increasing trend in the representation of mathematical models in publications in the epidemiological literature, from specialist journals of medicine, biology and mathematics to the highest impact generalist journals [ ] , showing the importance of interdisciplinary. their role in comparing, planning, implementing and evaluating various control programs is of major importance for public health decision makers. this interest has been reinforced by the recent examples of sars -severe acute respiratory syndrome -epidemic in and influenza pandemic in . although chronic diseases, such as cancer and heart diseases have been receiving more attention in developed countries, infectious diseases are still important and cause suffering and mortality in developing countries. these, remain a serious medical burden all around the world with million deaths per year estimated to be directly related to infectious diseases [ ] . the successful containment of the emerging diseases is not just linked to medical infrastructure but also on the capacity to recognize its transmission characteristics and apply optimal medical and logistic policies. public health often asks information such as [ ] : how many people will be infected, how many require hospitalization, what is the maximum number of people ill at a given time and how long will the epidemic last. as a result, it is necessary an ever-increasing capacity for a rapid response. education, vaccination campaigns, preventive drugs administration and surveillance programs, are all examples of prevention methods that authorities must consider for disease prevention. whenever the disease declares itself, the emergency interventions such as disinfectants, insecticide application, mechanical controls and quarantine measures must be considered. intervention strategies can be modelled with the goal of understanding how they will influence the disease's battle. as financial resources are limited, there is a pressing need to optimize investments for disease prevention and fight. traditionally, the study of disease dynamics has been focused on identifying the mechanisms responsible for epidemics but has taken little into account economic constraints in analyzing control strategies. on the other hand, economic models have given insight into optimal control under constraints imposed by limited resources, but they are frequently ignored by the spatial and temporal dynamics of the disease. therefore, progress requires a combination of epidemiological and economic factors for modelling what until here tended to remain separate. more recently, bioeconomic approaches to disease management have been advocated, since infectious diseases can be modelled thinking that the limited resources involved require trade-offs. finding the optimal strategy depends on the balance of economic and epidemiological parameters that reflect the nature of the host-pathogen system and the efficiency of the control method. the main goal of this thesis is to formulate epidemiological models, giving a special importance to dengue disease. moreover, it is our aim to frame the disease management question into an optimal control problem requiring the maximization/minimization of some objective function that depends on the infected individuals (biological issues) and control costs (economic issues), given some initial conditions. this way, will allow us to propose practical control measures to the authorities to assess and forecast the disease burden, such as an attack rate, morbidity, hospitalization and mortality. the thesis is composed by two parts. the first part, comprising chapters to , gives a mathematical background to support the original results presented in the second part, that is composed by chapters to . in chapter , the definition of optimal control problem, its possible versions and the adapted first order necessary conditions based on the pontryagin maximum principle are introduced. simple examples are chosen to exemplify the mathematical concepts. with the increasing of variables and complexity, optimal control problems can no longer be solved analytically and numerical methods are required. for this purpose, in chapter , direct and indirect methods are presented for their resolution. direct methods consist in the discretization of the optimal xii control problem, reducing it to a nonlinear constrained optimization problem. indirect methods are based on the pontryagin maximum principle, which in turn reduces the problem to a boundary value problem. for each approach, the software packages used in this thesis are described. in chapter , the basic building blocks of most epidemiological models are reviewed: sir (composed by susceptible-infected-recovered) and sis models (susceptible-infected-susceptible). for these, it is possible to develop some analytical results which can be useful in the understanding of simple epidemics. taking this as the basis, we discuss the dynamics of other compartmental models, bringing more complex realities, such as those with exposed or carrier classes. the second part of the thesis contains the original results and is focused on dengue fever disease. dengue is a vector borne disease, caused by a mosquito from the aedes family. it is mostly found in tropical and sub-tropical climates, mostly in urban areas. it can provoke a severe flu-like illness, and sometimes, in severe cases can be lethal. according to the world health organization about % of the world's population is now at risk [ ] . the main reasons for the choice of this particular disease are: • the importance of this disease around the world, as well as the challenges of its transmission features, prevention and control measures; • two portuguese-speaking countries (brazil and cape verde) have already experience with dengue, and in the last one, a first outbreak occurred during the development of this thesis, which allowed the development of a groundbreaking work; • the mosquito aedes aegypti, the main vector that transmits dengue, is already in portugal, on madeira island [ ] , which without carrying the disease, is considered a potential threat to public health and has been followed by the health authorities. in chapter information about the mosquito, disease symptoms, and measures to fight dengue are reported. an old problem related to dengue is revisited and solved by different approaches. finally, a numerical study is performed to compare different discretization schemes in order to analyze the best strategies for future implementations. in chapter , a seir+asei model is studied. the basic reproduction number and the equilibrium points are computed as well as their relationship with the local stability of the disease free equilibrium. this model implements a control measure: adulticide. a study to find the best strategies available to apply the insecticide is made. continuous and piecewise constant strategies are used involving the system of ordinary differential equations only or resorting to the optimal control theory. chapter is concerned with a sir+asi model that incorporates three controls: adulticide, larvicide and mechanical control. a detailed discussion on the effects of each control, individually or together, on the development of the disease is given. an analysis of the importance of each control in the decreasing of the basic reproduction number is given. these results are strengthened when the optimal strategy for the model is calculated. bioeconomic approaches, using distinct weights for the respective control costs and treatments for infected individuals have also been provided. in chapter simulations for a hypothetical vaccine for dengue are carried out. the features of the vaccine are unknown because the clinical trials are ongoing. using models with a new compartment for vaccinated individuals, perfect and imperfect vaccines are studied aiming the analysis of the repercussions of the vaccination process in the morbidity and/or eradication of the disease. then, an optimal control approach is studied, considering the vaccination not as a new compartment, but as a measure control in fighting the disease. finally, the main conclusions are reported and future directions of research are pointed out. xiv part i state of the art chapter the optimal control definition and its possible formulations are introduced, followed by some examples related to epidemiological models. the pontryagin maximum principle is presented with the aim of finding the best control policy. optimal control (oc) is the process of determining control and state trajectories for a dynamic system over a period of time in order to minimize a performance index [ ] . historically, oc is an extension of the calculus of variations. in the seventeenth century, the first formal results of calculus of variations can be found. johann bernoulli challenged other famous contemporary mathematicians -such as newton, leibniz, jacob bernoulli, l'hôpital and von tschirnhaus -with the brachistochrone problem: "if a small object moves under the influence of gravity, which part between two fixed points enables it to make the trip in the shortest time?" other specific problems were solved and a general mathematical theory was developed by euler and lagrange. the most fruitful applications of the calculus of variations have been to theoretical physics, particularly in connection with hamilton's principle or the principle of least action. early applications to economics appeared in the late s and early s by ross, evans, hottelling and ramsey, with further applications published occasionally thereafter [ ] . the generalization of the calculus of variations to optimal control theory was strongly motivated by military applications and has developed rapidly since . the decisive breakthrough was achieved by the russian mathematician lev s. pontryagin ( pontryagin ( - and his co-workers (v. g. boltyanskii, r. v. gamkrelidz and e. f. misshchenko) with the formulation and demonstration of the pontryagin maximum principle [ ] . this principle has provided research with suitable conditions for optimization problems with differential equations as constraints. the russian team generalized variational problems by separating control and state variables and admitting control constraints. in such problems, oc gives equivalents results, as one would have expected. however, the two approaches differ and the oc approach sometimes affords insight into a problem that might be less readily apparent through the calculus of variations. oc is also applied to problems where the calculus of variations is not convenient, such as those involving constraints on the derivatives of functions [ ] . the theory of oc brought new approaches to mathematics with dynamic programming. introduced by r. e. bellman, dynamic programming makes use of the principle of optimality and it is suitable for solving discrete problems, allowing for a significant reduction in the computation of the optimal controls (see [ ] ). it is also possible to obtain a continuous approach to the principle of optimality that leads to the solution of a partial differential equation called the hamilton-jacobi-bellman equation. this result allowed to bring new connections between the oc problem and the lyapunov stability theory. before the arrival of the computer, only fairly simple the oc problems could be solved. the arrival of the computer age enabled the application of oc theory and some methods to many complex problems. selected examples are as follows: • physical systems, such as stable performance of motors and machinery, robotics, optimal guidance of rockets [ , ] ; • aerospace, including driven problems, orbits transfers, development of satellite launchers and recoverable problems of atmospheric reentry [ , ] ; • economics and management, such as optimal exploitation of natural resources, energy policies, optimal investment of production strategies [ , ] ; • biology and medicine, as regulation of physiological functions, plants growth, infectious diseases, oncology, radiotherapy [ , , , ] . today, the oc theory is extensive and with several approaches. one can adjust controls in a system to achieve a goal, where the underlying system can include: ordinary differential equations, partial differential equations, discrete equations, stochastic differential equations, integro-difference equations, combination of discrete and continuous systems. in this work the goal is the oc theory of ordinary differential equations with time fixed. a typical oc problem requires a performance index or cost functional (j[x(t), u(t)]), a set of state variables (x(t) ∈ x), a set of control variables (u(t) ∈ u ) in a time t, with t ≤ t ≤ t f . the main goal consists in finding a piecewise continuous control u(t) and the associated state variable x(t) to maximize a given objective functional. the development of this chapter will be closely structured from lenhart and workman work [ ] . x(t f ) could be free, which means that the value of x(t f ) is unrestricted, or could be fixed, i.e, x(t f ) = x f . for our purposes, f and g will always be continuously differentiable functions in all three arguments. we assume that the control set u is a lebesgue measurable function. thus, as the control(s) will always be piecewise continuous, the associated states will always be piecewise differentiable. we have been focused on finding the maximum of a function. we can switch back and forth between maximization and minimization by simply negating the cost functional: an oc problem can be presented in different ways, but equivalent, depending on the purpose or the software to be used. there are three well known equivalent formulations to describe the oc problem, which are lagrange (already presented in previous section), mayer and bolza forms [ , ] . definition (bolza formulation). the bolza formulation of the oc problem can be defined as where φ is a continuously differentiable function. definition (mayer formulation). the mayer formulation of the oc problem can be defined as proof. ( ) ⇒ ( ) to get the proof, we formulate the bolza problem as one of lagrange, using an extended state vector. let (x(·), u(·)) an admissible pair for the problem ( . ) and let z(t) = (x a (t), . so, (z(·), u(·)) is an admissible pair for the lagrange problem thus, the value of the functionals in both formulations matches. ( ) ⇒ ( ) conversely, each admissible pair (z(·), u(·)) for the problem ( . ) corresponds the pair (x(·), u(·)), where x(·) is composed by the last component of z, admissible for the problem ( . ) and matching the respective values of the functionals. ( ) ⇒ ( ) for this statement we also need to use an extended state vector. let be thus we have (z(·), u(·)) an admissible pair for the following mayer problem: this way, the values of the functional for both formulations are the same. ( ) ⇒ ( ) conversely, to all admissible pair (z(·), u(·)) for the mayer problem ( . ) corresponds to an admissible pair (x(·), u(·)) for the bolza problem ( . ) , where x(·) consists in the last component of z(·). for the proof of the previous theorem it was not necessary to show that the lagrange problem is equivalent to the mayer formulation. however, in the second part of the thesis, and due to computational issues, some of the oc problems (usually presented in the lagrange form) will be converted into the equivalent mayer one. hence, using a standard procedure is possible to rewrite the cost functional (cf. [ ] ), augmenting the state vector with an extra component. so, the lagrange formulation ( . ) can be rewritten as max the necessary first order conditions to find the optimal control were developed by pontryagin and his co-workers. this result is considered as one of the most important results of mathematics in the th century. pontryagin introduced the idea of adjoint functions to append the differential equation to the objective functional. adjoint functions have a similar purpose as lagrange multipliers in multivariate calculus, which append constraints to the function of several variables to be maximized or minimized. definition (hamiltonian). let the previous oc problem considered in ( . ). the function h(t, x(t), u(t), λ(t)) = f (t, x(t), u(t)) + λ(t)g(t, x(t), u(t)) if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and proof. the proof of this theorem is quite technical and we opted to omit it. the original pontryagin's text [ ] or clarke's book [ ] are good references to find the proof. remark . the last condition, λ(t f ) = , called transversality condition, is only used when the oc problem does not have terminal value in the state variable, i.e., x(t f ) is free. this principle converted the problem of finding a control which maximizes the objective functional subject to the state ode and initial condition into the problem of optimizing the hamiltonian pointwise. as consequence, with this adjoint equation and hamiltonian, we have at u * for each t, namely, the hamiltonian has a critical point; usually this condition is called optimality condition. thus to find the necessary conditions, we do not need to calculate the integral in the objective functional, but only use the hamiltonian. here is presented a simple example to illustrate this principle. example (from [ ] ). consider the oc problem: the calculus of this oc problem can be done by steps. step -form the hamiltonian for the problem. the hamiltonian can be written as: step -write the adjoint differential equation, the optimality condition and transversality boundary condition (if necessary). try to eliminate u * by using the optimality equation h u = , i.e., solve for u * in terms of x * and λ. using the hamiltonian to find the differential equation of the adjoint λ, we obtained the optimality condition is given by in this way we obtain an expression for the oc: as the problem has just an initial condition for the state variable, it is necessary to calculate the transversality condition: λ( ) = . step -solve the set of two differential equations for x * and λ with the boundary conditions, replacing u * in the differential equations by the expression for the optimal control from the previous step. by the adjoint equation λ = − − λ and the transversality condition λ( ) = we have hence, the optimality condition leads to u * = −λ ⇔ u * = − e −t and the associated state is if the hamiltonian is linear in the control variable u, it can be difficult to calculate u * from the optimality equation, since ∂h ∂u would not contain u. specific ways of solving these kind of problems can be found in [ ] . until here we have showed necessary conditions to solve basic optimal control problems. now, it is important to study some conditions that can guarantee the existence of a finite objective functional value at the optimal control and state variables, based on [ , , , ] . the following is an example of a sufficient condition result. suppose that f (t, x, u) and g(t, x, u) are both continuously differentiable functions in their three arguments and concave in x and u. suppose u * is a control with associated state x * , and λ a piecewise differentiable function, such that u * , x * and λ together satisfy on t ≤ t ≤ t f : then for all controls u, we have proof. the proof of this theorem is available on [ ] . this result is not strong enough to guarantee that j(u * ) is finite. such results usually require some conditions on f and/or g. next theorem is an example of an existence result from [ ] . theorem . . . let the set of controls for problem ( . ) be lebesgue integrable functions on t ≤ t ≤ t f in r. suppose that f (t, x, u) is convex in u, and there exist constants c , c , c > , c and β > such that then there exists an optimal control u * maximizing j(u), with j(u * ) finite. proof. the proof of this theorem is available on [ ] . for a minimization problem, g would have a concave property and the inequality on f would be reversed. note that the necessary conditions developed to this point deal with piecewise continuous optimal controls, while this existence theorem guarantees an optimal control which is only lebesgue integrable. this disconnection can be overcome by extending the necessary conditions to lebesgue integrable functions [ , ] , but we did not expose this idea in the thesis. see the existence of oc results in [ ] . in some cases it is necessary, not only minimize (or maximize) terms over the entire time interval, but also minimize (or maximize) a function value at one particular point in time, specifically, the end of the time interval. there are some situations where the objective function must take into account the value of the state at the terminal time, e.g., the number of infected individuals at the final time in an epidemic model [ ] . definition (oc problem with payoff term). an oc problem with payoff term is in the form where φ(x(t f )) is a goal with respect to the final position or population level x(t f ). the term φ(x(t f )) is called payoff or salvage. using the pmp, adapted necessary conditions can be derived for this problem. proposition (necessary conditions). if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and proof. the proof of this result can be found in [ ] . a new example is given to illustrate this proposition. example (from [ ] ). let x(t) represent the number of tumor cells at time t, with exponential growth factor α, and u(t) the drug concentration. the aim is to minimize the number of tumor cells at the end of the treatment period and the accumulated harmful effects of the drug on the body. this problem is formulated as let us consider the hamiltonian the optimality condition is given by the adjoint condition is given by using the transversality condition λ(t f ) = (note that φ(s) = s, so φ (s) = ), we obtain the optimally state trajectory is (usingẋ = αx − u and x( ) = x ): many problems require bounds on the control to achieve a realistic solution. for example, the amount of drugs in the organism must be non-negative and it is necessary to impose a limit. for the last example, despite being simplistic, makes more sense to constraint the control as ≤ u ≤ . definition (oc with bounded control). an oc with bounded control can be written in the form where a, b are fixed real constants and a < b. to solve problems with bounds on the control, it is necessary to develop alternative necessary conditions. proposition (necessary conditions). if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and λ(t f ) = (transversality condition). by an adaptation of the pmp, the oc must satisfy (optimality condition): i.e., the maximization is over all admissible controls, andũ is obtained by the expression ∂h ∂u = . in particular, the optimal control u * maximizes h pointwise with respect to a ≤ u ≤ b. proof. the proof of this result can be found in [ ] . if we have a minimization problem, then u * is instead chosen to minimize h pointwise. this has the effect of reversing < and > in the first and third lines of optimality condition. remark . in some software packages there are no specific characterization for the bounds of the control. in those cases, and when the implementation allows, we can write in a compact way the optimal controlũ obtained without truncation, bounded by a and b: u * (t) = min{a, max{b,ũ}}. so far, we have only examined problems with one control and with one dependent state variable. often, it is necessary to consider more variables. definition (oc with several variables and several controls). an oc with n state variables, m control variables and a payoff function φ can be written in the form max u ,...,um x i (t ) = x i , i = , , . . . , n ( . ) where the functions f , g i are continuously differentiable in all variables. from now on, to simplify the notation, let x(t) = [x (t), . . . , x n (t)], u(t) = [u (t), . . . , u m (t)], x = [x , . . . , x n ], and g(t, x, u) = [g (t, x, u), . . . , g n (t, x, u)]. so, the previous problem can be rewritten in a compact way as x(t ) = x , i = , , . . . , n ( . ) using the same approach of the previous subsections, it is possible to derive generalized necessary conditions. proposition (necessary conditions). let u * be a vector of optimal control functions and x * be the vector of corresponding optimal state variables. with n states, we will need n adjoints, one for each state. there is a piecewise differentiable vector-valued function λ(t) = [λ (t), . . . , λ n (t)] , where each λ i is the adjoint variable corresponding to x i , and the hamiltonian is it is possible to find the variables satisfying identical optimality, adjoint and transversality conditions in each vector component. namely, u * maximizes h(t, x * , u, λ) with respect to u at each t, and u * , x * and λ satisfy x(t f )) for j = , . . . , n (transversality conditions) ∂h ∂u k = at u * k for k = , . . . , m (optimality conditions) by φ xj , it is meant the partial derivative in the x j component. note, if φ ≡ , then λ j (t f ) = for all j, as usual. similarly to the previous section, if bounds are placed on a control variable, a k ≤ u k ≤ b k (for k = , . . . , m), then the optimality condition is changed from ∂h ∂u k = to below, an optimal control problem related to rubella is presented. rubella, commonly known as german measles, is a most common in child age, caused by the rubella virus. children recover more quickly than adults, and can be very serious in pregnancy. the virus is contracted through the respiratory tract and has an incubation period of to weeks. the primary symptom of rubella virus infection is the appearance of a rash on the face which spreads to the trunk and limbs and usually fades after three days. other symptoms include low grade fever, swollen glands, joint pains, headache and conjunctivitis. it is presented now an optimal control problem to study the dynamics of rubella in china over three years, using a vaccination process (u) as a measure to control the disease (more details can be found in [ ] ). let x represent the susceptible population, x the proportion of population that is in the incubation period, x the proportion of population that is infected with rubella and x the rule that remains the population constant. the optimal control problem can be defined as: it is very difficult to solve analytically this problem. for most of the epidemiologic problems it is necessary to employ numerical methods. some of them will be described in the next chapter. in this chapter, some numerical approaches to solve a system of ordinary differential equations, such as shooting methods and multi-steps methods, are introduced. then, two distinct philosophies to solve oc problems are presented: indirect methods centered in the pmp and the direct ones focus on problem discretization solved by nonlinear optimization codes. a set of software packages used all over the thesis is summarily exposed. in the last decades the computational world has been developed in an amazing way. not only in hardware issues such as efficiency, memory capacity, speed, but also in terms of the software robustness. groundbreaking achievements in the field of numerical solution techniques for differential and integral equations have enabled the simulation of highly complex real world scenarios. this way, oc also won with these improvements and numerical methods and algorithms have evolved significantly. the next section concerns on the resolution of differential equations systems. a dynamic system is mathematically characterized by a set of ordinary differential equations (ode). specifically, the dynamics are described for t ≤ t ≤ t f , by a system of n odeṡ . . , y n (t), t) f (y (t), . . . , y n (t), t) . . . the problems of solving an ode are classified into initial value problems (ivp) and boundary value problems (bvp), depending on how the conditions at the endpoints of the domain are specified. all the conditions of an initial-value problem are specified at the initial point. on the other hand, the problem becomes a boundary-value problem if the conditions are needed for both initial and final points. there exist many numerical methods to solve initial value problems -such as euler, runge-kutta or adaptive methods -and boundary value problems, such as shooting methods. one can visualize the shooting method as the simplest technique for solving bvp. supposing it is desired to determine the initial angle of a cannon so that, when a cannonball is fired, it strikes a desired target. an initial guess is made for the angle of the cannon, and the cannon is fired. if the cannon does not hit the target, the angle is adjusted based on the amount of the miss and another cannon is fired. the process is repeated until the target is hit [ ] . suppose we want to find y(t ) = y such that y(t f ) = b. the shooting method can be summarized as follows [ ] : step . guess initial conditions x = y(t ); step . propagate the differential system equations from t to t f , i.e., shoot; step . evaluate the error in the boundary conditions c(x) = y(t f ) − b; step . use a nonlinear program to adjust the variables x to satisfy the constraints c(x) = , i.e., repeat steps - . despite its simplicity, from a practical standpoint, the shooting method is used only when the problem has a small number of variables. this method has a major disadvantage: a small change in the initial condition can produce a very large change in the final conditions. in order to overcome the numerical difficulties of the simple method, the multiple shooting method is presented. in a multiple shooting method, the time-interval [t , t f ] is divided into m − subintervals. then is applied over each subinterval [t i , t i+ ] with the initial values of the differential equations in the interior intervals being unknown that need to be determined. in order to enforce continuity, the following conditions are enforced at the interface of each subinterval: a scheme of the multiple shooting method is shown in figure with the multiple shooting approach the problem size is increased: additional variables and constraints are introduced for each shooting segment. in particular, the number of nonlinear variables and constraints for a multiple shooting application is n = n y (m − ), where n y is the number of dynamic variables y and m − is the number of segments [ ] . both shooting and multiple shooting methods require a good guess for initial conditions and the propagation of the shoots for problem of high dimension is not feasible. for this reason, other methods can be implemented, using initial value problems. the numerical solution of the ivp is fundamental to most optimal control methods. the problem can stand as follows: compute the value of y(t f ) for some value of t < t f that satisfies ( . ) with the known initial value y(t ) = y . numerical methods for solving the ode ivp are relatively mature in comparison to other fields in optimal control. it will be considered two methods: single-step and multiple-step methods. in both, the solution of the differential system at each step t k is sequentially obtained using current and/or previous information about the solution. in both cases, it is assumed that the time t = nh moves ahead in uniform steps of length h [ , ] . the most common single-step method is euler method. in this discretization scheme, if a differential equation is written likeẋ = f (x(t), t), is possible to make a convenient approximation of this: this approximation x n+ of x(t) at the point t n+ has an error of order h . clearly, there is a trade-off between accuracy and complexity of calculation which depends heavily on the chosen value for h. in general, as h is decreased the calculation takes longer but is more accurate. for many higher order systems it is very difficult to make euler approximation effective. for this reason more accurate and elaborate techniques were developed. one of these methods is the runge-kutta method. a runge-kutta method is a multiple-step method, where the solution at time t k+ is obtained from a defined set of previous values t j−k , . . . , t k and j is the number of steps. if a differential equation is written likeẋ = f (x(t), t), it is possible to make a convenient approximation of this, using the second order runge-kutta method or the fourth order runge-kutta method x n+ x n + h (k + k + k + k ) where this approximation x n+ of x(t) at the point t n+ has an error depending on h and h , for the runge-kutta methods of second and fourth order, respectively. numerical methods for solving oc problems date back to the s with bellman investigation. from that time to present, the complexity of methods and corresponding complexity and variety of applications has substantially increased [ ] . there are two major classes of numerical methods for solving oc problems: indirect methods and direct methods. the first ones, indirectly solve the problem by converting the optimal control problem to a boundary-value problem, using the pmp. on the other hand, in a direct method, the optimal solution is found by transcribing an infinite-dimensional optimization problem to a finite-dimensional optimization problem. in an indirect method, the pmp is used to determine the first-order optimality conditions of the original oc problem. the indirect approach leads to a multiple-point boundary-value problem that is solved to determine candidate optimal trajectories called extremals. for an indirect method it is necessary to explicitly get the adjoint equations, the control equations and all the transversality conditions, if there exist. notice that there is no correlation between the method used to solve the problem and its formulation: one may consider applying a multiple shooting method solution technique to either an indirect or a direct formulation. in the following subsection a numerical approach using the indirect method is presented. this method is described in a recent book by lenhart and workman [ ] and it is known as forwardbackward sweep method. the process begins with an initial guess on the control variable. then, the state equations are simultaneously solved forward in time and the adjoint equations are solved backward in time. the control is updated by inserting the new values of states and adjoints into its characterization, and the process is repeated until convergence occurs. considering x = (x , . . . , x n + ) and λ = (λ , . . . , λ n + ) the vector approximations for the state and the adjoint. the main idea of the algorithm is described as follows: step . make an initial guess for u over the interval ( u ≡ is almost always sufficient); step . using the initial condition x = x(t ) = a and the values for u, solve x forward in time according to its differential equation in the optimality system; step . using the transversality condition λ n + = λ(t f ) = and the values for u and x, solve λ backward in time according to its differential equation in the optimality system; step . update u by entering the new x and λ values into the characterization of the optimal control; step . verify convergence: if the variables are sufficiently close to the corresponding in the previous iteration, then output the current values as solutions, else return to step . for steps and , lenhart and workman used for the state and adjoint systems the runge-kutta fourth order procedure to make the discretization process. on the other hand, wang [ ] , applied the same philosophy but solving the differential equations with the solver ode for matlab. this solver is based on an explicit runge-kutta ( , ) formula, the dormand-prince pair. that means the numerical solver ode combines a fourth and a fifth order methods, both of which are similar to the classical fourth order runge-kutta method discussed above. these vary the step size, choosing it at each step an attempt to achieve the desired accuracy. therefore, the solver ode is suitable for a wide variety of initial value problems in practical applications. in general, ode is the best method to apply as a first attempt for most problems [ ] . let consider the open problem defined in chapter (example ) about rubella disease. with x(t) = (x (t), x (t), x (t), x (t)) and λ(t) = (λ (t), λ (t), λ (t), λ (t)), the hamiltonian of this problem can be written as using the pmp the optimal control problem can be studied with the state variableṡ with initial conditions x ( ) = . , x ( ) = . , x ( ) = . and x ( ) = and the adjoint variables: the optimal control is here it is only presented the main part of the code using the backward-forward sweep method with fourth order runge-kutta. the completed one can be found in the website [ ] . .. beta*(x (i)+h *m )*(x (i)+h *m )-( . *(u(i) + u(i+ )))*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); m = b-b*(p*(x (i)+h *m )+q*(x (i)+h *m ))-b*(x (i)+h *m )-... beta*(x (i)+h *m )*(x (i)+h *m )-( . *(u(i) + u(i+ )))*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); m = b-b*(p*(x (i)+h *m )+q*(x (i)+h *m ))-b*(x (i)+h *m )-... beta*(x (i)+h *m )*(x (i)+h *m )-u(i+ )*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); end for i = :m j = m + -i; n = lambda (j)*(b+u(j)+beta*x (j))-lambda (j)*beta*x (j); n = lambda (j)*b*p+lambda (j)*(e+b-p*b)-lambda (j)*e; n = -a+lambda (j)*(b*q+beta*x (j))-lambda (j)*beta*x (j)+lambda (j)*(g+b); n = b*lambda (j); n = (lambda (j) -h *n )*(b+u(j)+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- ))); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- )))+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); n = (lambda (j) -h *n )*(b+u(j)+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- ))); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- )))+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); n = (lambda (j) -h *n )*(b+u(j)+beta*x (j- ))-(lambda (j) -h *n )*beta*x (j- ); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*x (j- ))-... (lambda (j) -h *n )*beta*x (j- )+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); end u = min( . ,max( ,lambda .*x / )); the optimal curves for the states variables and optimal control are shown in figure there are several difficulties to overcome when an optimal control problem is solved by indirect methods. firstly, is necessary to calculate the hamiltonian, adjoint equations, optimality condition and transversality conditions. besides, the approach is not flexible, since each time a new problem is formulated, a new derivation is required. in contrast, a direct method does not require explicit derivation neither the necessary conditions. due to these practical difficulties with the indirect formulation, the main focus will be centered on the direct methods. this approach has been gaining popularity in numerical optimal control over the past three decades [ ] . a new family of numerical methods for dynamic optimization has emerged, referred to as direct methods. this development has been driven by the industrial need to solve large-scale optimization problems and it has also been supported by the rapidly increasing computational power. a direct method constructs a sequence of points x , x , . . . , x * such that the objective function in minimized and typical f (x ) > f (x ) > · · · > f (x * ). here the state and/or control are approximated using an appropriate function approximation (e.g., polynomial approximation or piecewise constant parameterization). simultaneously, the cost functional is approximated as a cost function. then, the coefficients of the function approximations are treated as optimization variables and the problem is reformulated to a standard nonlinear optimization problem (nlp) in the form: where c i , i ∈ e e c j , j ∈ i are the set of equality and inequality constraints, respectively. in fact, the nlp is easier to solve than the boundary-value problem, mainly due to the sparsity of the nlp and the many well-known software programs that can handle with this feature. as a result, the range of problems that can be solved via direct methods is significantly larger than the range of problems that can be solved via indirect methods. direct methods have become so popular these days that many people have written sophisticated software programs that employ these methods. here we present two types of codes/packages: specific solvers for oc problems and standard nlp solvers used after a discretization process. the oc-ode [ ] , optimal control of ordinary-differential equations, by matthias gerdts, is a collection of fortran routines for optimal control problems subject to ordinary differential equations. it uses an automatic direct discretization method for the transformation of the oc problem into a finite-dimensional nlp. oc-ode includes procedures for numerical adjoint estimation and sensitivity analysis. considering the same problem (example ), here is the main part of the code in oc-ode. the completed one can be found in the website [ ] . the achieved solution is similar to the indirect approach, therefore we will not present it. the dotcvp [ ] , dynamic optimization toolbox with vector control parametrization is a dynamic optimization toolbox for matlab. the toolbox provides environment for a fortran compiler to create the '.dll' files of the ode, jacobian, and sensitivities. however, a fortran compiler has to be installed in a matlab environment. the toolbox uses the control vector parametrization approach for the calculation of the optimal control profiles, giving a piecewise solution for the control. the oc problem has to be defined in mayer form. for solving the nlp, the user can choose several deterministic solvers -ipopt, fmincon, fsqp -or stochastic solvers -de, sres. the modified sundials tool [ ] is used for solving the ivp and for the gradients and jacobian automatic generation. forward integration of the ode system is ensured by cvodes, a part of sundials, which is able to perform the simultaneous or staggered sensitivity analysis too. the ivp problem can be solved with the newton or functional iteration module and with the adams or bdf linear multistep method. note that the sensitivity equations are analytically provided and the error control strategy for the sensitivity variables could be enabled. dotcvp has a user friendly graphical interface (gui). considering the same problem (example ), here is a part of the code used in dotcvp. the completed one can be found in the website [ ] . the solution, despite being piecewise continuous, follows the curve obtained by the previous programs. functional for non-stiff problems data.odes.linearsolver with the gui interface this method was the preferred to be tested, due to its simple way to implement the code. in neos [ ] platform there is a large set of software packages. neos is considered as the state of the art in optimization. one recent solver is muscod-ii [ ] (multiple shooting code for optimal control) for the solution of mixed integer nonlinear ode or dae constrained optimal control problems in an extended ampl format. ampl [ ] is a modelling language for mathematical programming and was created by fourer, gay and kernighan. the modelling languages organize and automate the tasks of modelling, which can handle a large volume of data and, moreover, can be used in machines and independent solvers, allowing the user to concentrate on the model instead of the methodology to reach solution. however, the ampl modelling language itself does not allow the formulation of differential equations. hence, the taco toolkit has been designed to implement a small set of extensions for easy and convenient modeling of optimal control problems in ampl, without the need for explicit encoding of discretization schemes. both the taco toolkit and the neos interface to muscod-ii are still under development. probably for this reason, the example could not be solved by this software which crashed after some runs. anyway, we opted to also put the code, for the same example that is being used, to show the differences of modelling language used in each program. include optimalcontrol.mod; var t ; var x , >= <= ; var x , >= <= ; var x , >= <= ; var x , >= <= ; var u >= , <= . suffix type "u "; the three nonlinear optimization software packages presented here, were used through the neos platform with codes formulated in ampl. the ipopt [ ] , interior point optimizer, is a software package for large-scale nonlinear optimization. it is written in fortran and c. ipopt implements a primal-dual interior point method and uses a line search strategy based on filter method. ipopt can be used from various modeling environments. ipopt is designed to exploit st and nd derivative information if provided, usually via automatic differentiation routines in modeling environments such as ampl. if no hessians are provided, ipopt will approximate them using a quasi-newton methods, specifically a bfgs update. continuing with example the ampl code is shown for ipopt. the euler discretization was the selected. indeed, this code can also be implemented in other nonlinear software packages available in neos platform, reason why the code for the next two software packages will not be shown. the full version can be found on the website [ ] . , short for "nonlinear interior point trust region optimization", was created primarily by richard waltz, jorge nocedal, todd plantenga and richard byrd. it was introduced in as a derivative of academic research at northwestern, and has undergone continual improvement since then. knitro is also a software for solving large scale mathematical optimization problems based mainly on the two interior point (ip) methods and one active set algorithm. knitro is specialized for nonlinear optimization, but also solves linear programming problems, quadratic programming problems, and systems of nonlinear equations. the unknowns in these problems must be continuous variables in continuous functions; however, functions can be convex or nonconvex. the code also provides a multistart option for promoting the computation of the global minimum. this software was tested through the neos platform. snopt [ ] , by philip gill, walter murray and michael saunders, is a software package for solving large-scale optimization problems (linear and nonlinear programs). it is specially effective for nonlinear problems whose functions and gradients are expensive to evaluate. the functions should be smooth but do not need to be convex. snopt is implemented in fortran and distributed as source code. it uses the sqp (sequential quadratic programming) philosophy, with an augmented lagrangian approach combining a trust region approach adapted to handle the bound constraints. snopt is also available in neos platform. choosing a method for solving an oc problem depends largely on the type of problem to be solved and the amount of time that can be invested in coding. an indirect shooting method has the advantage of being simple to understand and produces highly accurate solutions when it converges [ ] . the accuracy and robustness of a direct method is highly dependent upon the method used. nevertheless, it is easier to formulate highly complex problems in a direct way and can be used standard nlp solvers as an extra advantage. this last feature has the benefit of converging with poor initial guesses and being extremely computationally efficient since most of the solvers exploit the sparsity of the derivatives in the constraints and objective function. in the next chapter the basic concepts from epidemiology are provided, in order to formulate/implement more complex oc problems in the health area. in this chapter, the simplest epidemiologic models composed by mutually exclusive compartments are introduced. based on the models sis (susceptible-infected-susceptible) and sir (susceptible-infected-recovered), other models are presented introducing new issues related to maternal immunity or the latent period, fitting the features to distinct diseases. illustrative examples are presented, with diseases that can be described by each model. the basic reproduction number is calculated and presented as a threshold value for the eradication or the persistence of the disease in a population. in the th century, occurred one of most famous epidemic events: the black death. it killed approximately one third of the european population. from - , twenty to forty percent of the world's population suffered from the spanish flu, the most severe pandemic in history. in , the united nations promoted an ambitious agreement between the countries forecasting that in the year infectious diseases would be eradicated. this conjecture failed, mainly due to the assumption that the microorganisms were biologically stationary and consequently they were not modified and became resistant to the medicines. besides, the improvements in the transportation allowing for a faster movement of individuals and the population growing especially in developing countries, led to the appearance of new diseases and the resurgence of old ones in distinct places. nowadays, aids is the most scrutinized. in there were an estimated . million sufferers worldwide, and . million deaths with over three quarters of these occurring in sub-saharan africa [ ] . epidemiology -the study of patterns of diseases including those which are non-communicable of infections in population -has become more relevant and indispensable in the development of new models and explanations for the outbreaks, namely due to their propagation and causes. in epidemiology, an infection is said to be endemic in a population when it is maintained in the population without the need for external inputs. an epidemic occurs when new cases of a certain disease appears, in a given human population during a given period, and then essentially disappears. there are several types of diseases, depending on their type of transmission mechanism, of which stand out: • bacteria, which do not confer immunity against the reinfection and frequently produce harmful toxins to the host; in case of infection, the antibiotics are usually efficient (examples: tuberculosis, meningitis, gonorrhea, syphilis, tetanus); • viral agents, that confer immunity against reinfection; here the antibiotics do not produce effects and usually it is hoped that the immune system of the host responds to an infection by the virus or it will be necessary to take antiviral drugs that retard the multiplication of the virus (examples influenza, chicken pox, measles, rubella, mumps, hiv / aids, smallpox); • vectors, that are usually mosquitoes or ticks and are infected by humans and then transmit the disease to other humans (examples: malaria, yellow fever, dengue, chikungunya). the transmission can happen in a direct or indirect way. the direct transmission of a disease can happen by physical proximity (such as sneezing, coughing, kissing, sexual contact) or even by a specific parasite that penetrates the host through ingestion or the skin. the indirect transmission involves the vectors that are intermediaries or carriers of the infection. in most of the cases, the direct and indirect transmission of the disease happens between the member that coexists in the host population; this is called the horizontal transmission. when the direct transmission occurs from one ascendent to a descendent not yet born (egg or embryo) it is said that vertical transmission happens [ ] . when formulating a model for a particular disease, we should make a trade-off between simple models -that omit several details and generally are used for specific situations in a short time, but have the disadvantage of possibly being naive and unrealistic -and more complex models, with more details and more realistic, but generally more difficult to solve or could contain parameters which their estimates cannot be obtained. choosing the most appropriated model depends on the precision or generality required, the available data, and the time frame in which the results are needed. by definition, all models are "wrong", in the sense that even the most complex will make some simplifying assumptions. it is, therefore, difficult to definitively express which model is right, though naturally we are interested in developing models that capture the essential features of a system. the art of epidemiological modelling is to make suitable choices in the model formulation making it as simple as possible and yet suitable for the question being considered [ ] . mathematical models are a simplified representation of how an infection spreads across a population over time, and generally come in two forms: stochastic and deterministic models. the first ones, employ randomness, with variables being described by probability distributions. deterministic models split the population into subclasses, and an ode with respect to time is formulated for each. the state variable are determined using parameters and initial conditions. the main focus in this chapter will be the deterministic models, neglecting the others. most epidemic models are based on dividing the population into a small number of compartments. each containing individuals that are identical in terms of their status with respect to the disease in question. here are some of the main compartments that a model can contain. • passive immune (m ): is composed by newborns that are temporary passively immune due to antibodies transferred by their mothers; • susceptible (s): is the class of individuals who are susceptible to infection; this can include the passively immune once they lose their immunity or, more commonly, any newborn infant whose mother has never been infected and therefore has not passed on any immunity; • exposed or latent (e): compartment referred to the individuals that despite infected, do not exhibit obvious signs of infection and the abundance of the pathogen may be too low to allow further transmission; • infected (i): in this class, the level of parasite is sufficiently large within the host and there is potential in transmitting the infection to other susceptible individuals; • recovered or resistant (r): includes all individuals who have been infected and have recovered. the choice of which compartments to include in a model depends on the characteristics of the particular disease being modelled and the purpose of the model. the exposed compartment is sometimes neglected, when the latent period is considered very short. besides, the compartment of the recovered individuals cannot always be considered since there are diseases where the host has never became resistent. acronyms for epidemiology models are often based on the flow patterns between the compartments such as mseir, mseirs, seir, seirs, sir, sirs, sei, seis, si, sis. there are three commonly used threshold values in epidemiology: r , σ and r. the most common and probably the most important is the basic reproduction number [ , , ] . definition (basic reproduction number). the basic reproduction number, denoted by r , is defined as the average number of secondary infections that occurs when one infective is introduced into a completely susceptible population. this threshold, r , is a famous result due to kermack and mckendrick [ ] and is referred to as the "threshold phenomenon", giving a borderline between a persistence or a disease death. r it is also called the basic reproduction ratio or basic reproductive rate. definition (contact number). the contact number, σ is the average number of adequate contacts of a typical infective during the infectious period. an adequate contact is one that is sufficient for transmission, if the individual contacted by the susceptible is an infective. it is implicitly assumed that the infected outsider is in the host population for the entire infectious period and mixes with the host population in exactly the same way that a population native would mix. definition (replacement number). the replacement number, r, is the average number of secondary infections produced by a typical infective during the entire period of infectiousness. note that the replacement number r changes as a function of time t as the disease evolves after the initial invasion. these three quantities r , σ and r are all equal at the beginning of the spreading of an infectious disease when the entire population (except the infective invader) is susceptible. r is only defined at the time of invasion, whereas σ and r are defined at all times. the replacement number r is the actual number of secondary cases from a typical infective, so that after the infection has invaded a population and everyone is no longer susceptible, r is always less than the basic reproduction number r . also after the invasion, the susceptible fraction is less than one, and as such not all adequate contacts result in a new case. thus the replacement number r is always less than the contact number σ after the invasion [ ] . combining these results leads to note that r = σ for most models, and σ > r after the invasion for all models. for the models throughout this study the basic reproduction number, r , will be applied. when r < the disease cannot invade the population and the infection will die out over a period of time. the amount of time this will take generally depends on how small r is. when invasion is possible and infection can spread through the population. generally, the larger the value of r the more severe, and possibly widespread, the epidemic will be [ ] . in table . are some example diseases with their estimated basic reproduction number. due to differences in demographic rates, rural-urban gradients, and contact structure, different human populations may be associated with different values of r for the same disease [ ] . in the next section some of the epidemiologic models will be presented. numerous infectious diseases confer no long-lasting immunity. the sis models are suitable for some bacterial agent diseases like meningitis, sexually transmitted diseases such as gonorrhea and for protozoan agent diseases where malaria and the sleeping sickness are good examples. for these diseases, an individual can be infected multiple times throughout their lives, with no apparent immunity. here, recovery from infection is followed by an instant return to the susceptible compartment. throughout this chapter we will consider that population is constant, neglecting the tourism and immigration factors. also it is considered that the population is homogeneously mixed, which means that every individual interacts with another at the same level and therefore all individuals have the same risk of contracting the disease. the number of individuals in each compartment must be integer, but if the population size n is sufficiently large, it is possible to treat s and i as continuous variables. calculating the proportion of these compartments, varying from to , it is considered that the total population is constant over time, i.e., = s + i. the compartment changes are expressed by a system of differential equations. the sis model can be mathematically represented as follows. definition (sis model). the sis model can be formulated as subject to initial conditions s( ) > and i( ) ≥ . it is considered β the transmission rate (per capita) and γ the recovery rate, so the mean infectious period is /γ. the vital dynamics (births and deaths) were not considered, but a similar model can be constructed with these effects [ ] . despite this lack of susceptible births, the disease can still persist because the recovery of infected individuals replenishes the susceptible class and will guarantee the long-term persistence of the disease. remark . the si model is a particular case of the sis model when the recovery rate (γ) is null. a new infected individual is expected to infect another at a transmission rate β, during γ that is the infectious period, so the expected basic reproduction number is trachoma is an infectious disease causing a characteristic roughening of the inner surface of the eyelids. also called granular conjunctivitis or egyptian ophthalmia, it is the leading cause of infectious blindness in the world. adapting a model from [ ] , and using β = . as transmission rate and the recovery rate γ = . , we have the basic reproduction number r approximately . and the following representation of the state variables (figure . ). the sir model was initially studied in depth by kermack and mckendrick and categorizes hosts within a population as susceptible, infected and recovered [ ] . it captures the dynamics of acute infections that confers lifelong immunity once recovered. diseases where individuals acquire permanent immunity, and for which this model may be applied, include measles, smallpox, chickenpox, mumps, typhoid fever and diphtheria. once again we will consider the total population size constant, i.e., = s + i + r. two cases will be studied, distinguished by the inclusion or exclusion of demographic factors. having compartmentalized the population, we now need a set of equations that specify how the sizes of compartments change over time. definition (sir model without demography). the sir model, excluding births and deaths, can be defined as in addition, the transmission rate, per capita, is β and the recovery rate is γ. since population is constant and as r does not appear in the first two differential equations, most of the times the last equation is omitted, indeed, r(t) = − s(t) − i(t). by assuming equations from ( . ) is possible to notice that ds dt then a newly introduced infected individual can be expected to infect other people at the rate β during the expected infectious period /γ. thus, this first infective individual can be expected to infect consider an epidemic of influenza in a british boarding school in early [ ] . three boys were reported to the school infirmary with the typical symptoms of influenza. over the next few days, a very large fraction of the boys in the school had contact with the infection. within two weeks, the infection had become extinguished. the best fit parameters yield an estimated active infectious period of /γ = . days and a mean transmission rate β = . per day. therefore, the estimated r is . . figure . represents the dynamics of the three state variables. it was presented a sir model given the assumption that the time scale of disease spread was sufficiently fast that births and deaths can be neglected. in the next subsection we explore the long-term persistence and endemic dynamics of an infectious disease. the simplest and most common way of introducing demography into the sir model is to assume there is a natural host lifespan, /µ years. then, the rate at which individuals, at any epidemiological compartment, suffer natural mortality is given by µ. it is important to emphasize that this factor is independent of the disease and is not intended to reflect the pathogenicity of the infectious agent. historically, it has been assumed that µ also represents the population's crude birth rate, thus ensuring that total population size does not change through time, or in other words, ds dt + di dt + dr dt = . putting all these assumptions together, we have a new definition. definition (sir model with demography). the sir model, including births and deaths, can be defined as the epidemiological scheme is in figure . . it is important to introduce the r expression for this model. the parameter β represents the trans-mission rate per infective and the negative terms in the equation tell us that each individual spends an average γ+µ time units in this class. therefore, if we assume the entire population is susceptible, then the average number of new infectious per infectious individual is determined by the inclusion of demographic dynamics may allow a disease to die out or persist in a population in the long term. for this reason it is important to explore what happens when the system is at equilibrium. definition (equilibrium points). a model defined sir has an equilibrium point, if a triple e * = (s * , i * , r * ) satisfies the following system: if the equilibrium point has the infectious component equal to zero (i * = ), this means that the pathogen suffered extinction and e * is called disease free equilibrium (dfe). if i * > the disease persist in the population and e * is called endemic equilibrium (ee). with some calculations and algebraic manipulations, it is possible to obtain two equilibria for the system ( . ): dfe: when r < , each infected individual produces, on average, less than one new infected individual, and therefore, predictable that the infection will be cleared from the population. if r > , the pathogen is able to invade the susceptible population [ , ] . it is possible to prove that for the endemic equilibrium to be stable, r must be greater than one, otherwise the disease free equilibrium is stable. more detailed information about local and global stability of the equilibrium point can be found in [ , , , ] . this threshold behavior is very useful, once we can determine which control measures, and at what magnitude, would be most effective in reducing r below one, providing important guidance for public health initiatives. the sir model below, figure . , is plotted using similar parameters and initial conditions, except the transmission rate β (adapted from [ ] ). it is shown that in case (a), using β = , the basic reproduction number is greater than one. with demographic effects could have damped oscillations with a decreasing amplitude, in the ee direction. in case (b), with β = , we obtain r < and the system tends to go to a dfe. the next three sections present a brief description of other possible refinements of the basic models sis and sir. in the seir case, the transmission process occurs with an initial inoculation with a very small number of pathogens. then, during a period of time the pathogen reproduces rapidly within the host, relatively unchallenged by the immune system. during this stage, pathogen abundance is too low for active transmission to other susceptible host, and yet the pathogen is present. the time in this stage is very difficult to quantify, since there is no symptomatic features of the disease. it is called the latent or exposed period. assuming the average duration of the latent period is given by ν , the seir model can be described as follow. definition (seir model). the seir model is formulated as the parameter β and γ were defined in the previous section. the epidemiological scheme for seir model is presented in figure this threshold is the product of the contact rate β per unit time, the average infectious period adjusts to the population growth of γ+µ , and the fraction ν ν+µ of exposed people surviving the latent class e. finding steady states of the system, we obtain the following equilibrium points: although the sir and seir models behave similarly at equilibrium, when the parameters are suitably adapted, the seir model has a slower growth rate after pathogen invasion. this is due to the fact that individuals need to stay some time in the exposed class before their contribution in the disease transmission chain [ ] . an infected or vaccinated mother transfers some antibodies across the placenta to her fetus, so that the newborn infant has temporary passive immunity to an infection. since the infant can not produce new antibodies, when these passive antibodies are gone at a rate δ, the baby passes from the immune state m to the susceptible state s. some childhood diseases have this feature. the birth rate µs into the susceptible class of size s corresponds to newborns whose mothers are susceptible, and the other newborns µ( − s) enter passively immune class of size m , since their mothers were infected or had some type of immunity [ ] . the transfer diagram for the mseir model is shown in the mseir is also composed by a system of differential equations. definition (mseir model). the seir model can be described as thus, the basic reproduction number is equal to previous model seir, once the m compartment does not affect the transmission chain of the disease: the equations ( . ) always have a dfe given by there are several models with different epidemiological states, such as mseirs, seirs, sei, si, sirs, depending on the specific features and the level of detail that is wished to introduce in the model. these models are similar to the previous ones presented. other epidemiological models can be studied using more compartments such as quarantine-isolation (q), treatment (t ), carrier (c) or vaccination (v ). besides, most of the populations can be subdivided into different groups (sex, age, health weaknesses, ...), depending upon characteristics that may influence the risk of catching and/or transmitting an infection. more details and examples can be found in [ , , , ] . other diseases can be caught and transmitted by numerous hosts or even is required a second different population to complete the transmission cycle, such as vector borne-diseases, using double models. this last case will be explored in the second part of the thesis. in cases that include multiple compartments of infected individuals, in which vital and epidemiological parameters depend on factors as stage of the disease, spatial position, age, behavior, multigroups, the next generation method is the more generalized approach to calculate r . the definition of r has more than one possible interpretation, depending on the field (ecology, demography or epidemiology) and there exist distinct methods and estimations to calculate this threshold. the next generation method, introduced by diekmann et al. [ ] , defines r as the spectral radius of the next generator operator. the formation of the operator involves the determination of two compartments, infected and non-infected from the model. recent examples of this method are given in [ , , , ] . let us assume that there are n compartments of which m are infected. we defined the vector x = (x , . . . , x n ) t , where x i ≥ denotes the number of proportion of individuals in the ith compartment. for clarity we sort the compartments so that the first m compartments correspond to infected individuals. the distinction between infected and uninfected compartments must be determined from the epidemiological interpretation, and not by the mathematical expressions. it is necessary to define the set the disease transmission model consists of nonnegative initial conditions together with the following system of equations:ẋ note that f i should include only infections that are newly arising, but does not include terms which describe the transfer of infectious individuals from one infected compartment to another. let us consider the following assumptions: (a ) if f(x) is set to zero, then all eigenvalues of df (x ) have negative real parts and df (x ) is the derivative ∂fi ∂xj evaluated at the dfe x . assuming that f i and v i meet the assumptions above, we can form the next generation matrix f v − from matrices of partial derivatives of f i and v i . specially, where i, j = , . . . , m and x is the dfe. the entries of f v − give the rate at which infected individuals in x j produce new infections in x i , times the average length of time an individual spends on a single visit to compartment j. definition (basic reproduction number using the next generator operator). the basic reproduction number is given by where ρ denotes the spectral radius (dominant eigenvalue) of the matrix f v − . the following theorem states that r is a threshold parameter for the stability of the dfe. proof. the proof of this theorem can be found in [ ] . consider a simple model seit for tuberculosis with a treated compartment (adapted from [ ] ). tuberculosis, is a common, and in many cases lethal, infectious disease caused by various strains of mycobacteria. tuberculosis typically attacks the lungs but can also affect other parts of the body. most infections are asymptomatic and latent, but about one in ten latent infections eventually progresses to active disease which, if left untreated, kills more than % of those so infected. in this model a constant population is considered where n = s + e + i + t . exposed individuals progress to the infectious compartment at a rate ν. the treatment rates are r for exposed individuals and r for infectious individuals. however only a fraction q of the treatments of infectious individuals are successful. unsuccessfully treated infectious individuals re-enter the exposed compartment ( − q). the dynamics are illustrated in figure . and the differential equations are the following: e and i, which gives m = , and we will construct the matrices f and v only related to these compartments. let this way, epidemiological models can be understood as a framework to explain the mechanisms of the disease procedures and test ideas to implement control measures. r is a key concept, used as a threshold parameter to predict where an infection will spread and how these controls can be effective. in the second part of the thesis, the epidemiological models will be used for studying dengue disease. applying oc theory, the repercussions of several controls in the development of the disease will be analyzed. during the last decades, the global prevalence of dengue progressed dramatically. in this chapter, dengue details are given, such as disease symptoms, transmission and epidemiological trends. an old oc model for dengue is revisited, as a first approach to the disease. due to the software robustness improvements and the higher computational capacity, a better solution for this problem is proposed. in order to study different discretization schemes for an oc problem, taking into account time performance and resources used, some numerical simulations are made using euler and runge-kutta methods. the origins of the word dengue are not clear. some researchers think that it is derived from the swahili phrase "ka-dinga pepo", meaning "cramp-like seizure caused by an evil spirit". the first recognized dengue epidemics occurred almost simultaneously in asia, africa, and north america in the s, shortly after the identification and naming of the disease in [ ] . dengue transcends international borders and can be found in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas. dengue is a disease which is now endemic in more than one hundred countries of africa, america, asia and the western pacific. in figure . it is possible to see the areas that in have more surveillance. nevertheless, some studies have indicated that countries with a mild climate, such as in the mediterranean, are at risk due to future climate conditions that may be favorable to this kind of disease [ ] . in europe there are no registered cases, but the main vector of the disease is already in the old continent and has been followed on madeira island [ , ] . this risk may be aggravated further due to climate changes and to the globalization, as a consequence of the huge volume of international tourism and trade [ ] . travelers play an essential role in the global epidemiology: they act as viremic travelers, carrying the disease into areas where mosquitos can transmit the infection. dengue is a vector-borne disease transmitted from an infected human to a female aedes mosquito by a bite. then, the mosquito, that needs regular meals of blood to feed their eggs, bites a potential healthy human and transmits the disease making it a cycle. there are two forms of dengue: dengue fever (df) and dengue hemorrhagic fever (dhf). the first one is characterized by a sudden high fever without respiratory symptoms, accompanied by intense headaches, painful joints and muscles and lasts between three to seven days. humans may only transmit the virus during the febrile stage [ ] . dhf initially exhibits a similar, if more severe pathology as df, but deviates from the classic pattern at the end of the febrile stage [ ] . the hemorrhagic form has an additionally bleeding from the nose, mouth and gums or skin bruising, nausea, vomiting and fainting due to low blood pressure by fluid leakage. it usually lasts between two to three days and can lead to death [ ] . nowadays, dengue is the mosquito-borne infection that has become a major international public health concern. according to the world health organization (who), to million dengue fever infections occur yearly, including dengue hemorrhagic fever cases and deaths, mostly among children [ ] . there are four distinct, but closely related, viruses that cause dengue. the four serotypes, named den- to den- , belong to the flavivirus family, but they are antigenically distinct. recovery from infection by one virus provides lifelong immunity against that virus but confers only partial and transient protection against subsequent infection by the other three viruses. there is good evidence that a sequential infection increases the risk of developing dhf [ ] . unfortunately, there is no specific effective treatment for dengue. activities, such as triage and management, are critical in determining the clinical outcome of dengue. a rapid and efficient front-line response not only reduces the number of unnecessary hospital admissions but also saves lives. although up until now there is no effective and safe vaccine for dengue, a number of candidates are undergoing various phases of clinical trials [ ] . with four closely related viruses that can cause the disease, there is a need for a vaccine that would immunize against all four types to be effective. the main difficulty in the vaccine production is that there is a limited understanding of how the disease typically behaves and how the virus interacts with the immune system. another challenge is that some studies show that some secondary dengue infection can leave to dhf, and theoretically a vaccine could be a potential cause of severe disease if a solid immunity is not established against the four serotypes. research to develop a vaccine is ongoing and the incentives to study the mechanism of protective immunity are gaining more support, now that the number of outbreaks around the world is increasing [ ] . the spread of dengue is attributed to the geographic expansion of the mosquitoes responsible for the disease: aedes aegypti and aedes albopictus [ ] . due to its higher interaction with humans and its urban behavior, the first mosquito is considered the major responsible for dengue transmission and, our attention will be focused on it. aedes aegypti, in figure . , is an insect species closely associated with humans and their dwellings, thriving in crowded cities and biting primarily during the day. humans not only provide blood meals for mosquitoes, but also nutrients needed for them to reproduce through water-holding containers, in and around their homes. in urban areas, aedes mosquitoes breed on water collections in artificial containers such as cans, plastic cups, used tires, broken bottles and flower pots. with increasing urbanization, crowded cities, poor sanitation and lack of hygiene, environmental conditions foster the spread of the disease that, even in the absence of fatal forms, breed significant economic and social costs (absenteeism, immobilization, debilitation and medication) [ ] . dengue is spread only by adult females that require a blood meal for the development of their eggs, whereas male mosquitoes feed on fruit nectar and other sources of sugar. in this process the female acquire the virus while feeding on the blood of an infected person. after virus incubation from eight to twelve days (extrinsic period), an infected mosquito is capable, during probing and blood feeding, of transmitting the virus for the rest of its life to susceptible humans, and the intrinsic period for humans varies from to days. the life cycle of a mosquito has four distinct stages: egg, larva, pupa and adult, as it is possible to see in figure . . in the case of aedes aegypti, the first three stages take place in or near water whilst air is the medium for the adult stage [ ] . female mosquitoes lay their eggs, but usually do not lay them all at once: it releases them in different places, increasing the probability of new births [ ] . the eggs of aedes aegypti can resist to droughts and low temperatures for up to one year. although the hatching of mature eggs may occur spontaneously at any time, this is greatly stimulated by flooding. larvae hatch when water inundates the eggs as a result of rains or an addition of water by people. in the following days, the larvae will feed on microorganisms and particulate organic matter. when the larva has acquired enough energy and size, metamorphosis is done, changing the larva into pupa. pupae do not feed: they just change in form until the adult body is formed. the newly formed adult emerges from the water after breaking the pupal skin. the entire life cycle, from the aquatic phase (eggs, larvae, pupae) to the adult phase, lasts from to days at room temperature, depending on the level of feeding [ ] . the adult stage of the mosquito is considered to last an average of eleven days in an urban environment, reaching up to days in laboratory environment. studies suggest that most female mosquitoes may spend their lifetime in or around the houses where they emerge as adults. this means that people, rather than mosquitoes, rapidly move the virus within and between communities. aedes aegypti is one of the most efficient vectors for arboviruses because it is highly anthropophilic, frequently bites several times before complete oogenesis [ ] . the extent of dengue transmission is determined by a wide variety of factors: the level of herd immunity in the human population to circulating virus serotype(s); virulence characteristics of the viral strain; survival, feeding behavior, and abundance of aedes aegypti; climate; and human density, distribution, and movement [ ] . it is very difficult to control or eliminate aedes aegypti mosquitoes because they are highly resilient, quickly adapting to changes in the environment and they have the ability to rapidly bounce back to initial numbers after disturbances resulting from natural phenomena (e.g., droughts) or human interventions (e.g., control measures). we can safely expect that transmission thresholds will vary depending on a range of factors. primary prevention of dengue resides mainly in mosquito control. there are two primary methods: larval control and adult mosquito control, depending on the intended target [ ] . larvicide treatment is done through long-lasting chemical in order to kill larvae and preferably have who clearance for use in drinking water [ ] . the application of adulticides can have a powerful impact on the abundance of adult mosquito vector. however, the efficacy is often constrained by the difficulty in achieving sufficiently high coverage of resting surfaces [ ] . this is the most common measure. however the long term use of adulticide has several risks: the resistance of the mosquito to the product reducing its efficacy, the killing of other species that live in the same habitat and has also been linked to numerous adverse health effects including the worsening of asthma and respiratory problems. larvicide treatment is an effective way to control the vector larvae, together with mechanical control, which is related to educational campaigns. the mechanical control must be done by both public health officials and residents in affected area. the participation of the entire population is essential in removing still water from domestic recipients and eliminating possible breeding sites [ ] . the most recent approach for fighting the disease is biological control. it is a natural process of population regulation through natural enemies. there are techniques that combine some parasites that kill partially the larval population; however there are some operational resistance, because there is lack of expertise in producing these type of parasites and there are some cultural objections in introducing something in the water for human consumption [ ] . another way of insect control is to change the reproduction process, releasing sterile insects. this technique, named as sterile insect technique, consists in releasing sterile insects in natural environment, so that the result of mating produces the non-viability of the eggs, and thus can lead to drastic reduction of the specie. this way of control, shows two types of inconvenience: it is expensive to produce and release insects, and can be confronted with social objections, because an uninformed population could not correctly understand the addition of insects as a good solution [ , , ] . mathematical modeling became an interesting tool for understanding epidemiological diseases and for proposing effective strategies in fighting them. a set of mathematical models have been developed in literature to gain insights into the transmission dynamics of dengue in a community. while zeng and velasco-hérnandez [ ] investigate the competitive exclusion principle in a two-strain dengue model, chowell et al. [ ] estimates the basic reproduction number for dengue using spatial epidemic data. in [ ] the author studies the spread of dengue thought statistical analysis, while in tewa et al. [ ] global asymptotic stability of the equilibrium of a single-strain dengue model is established. the control of the mosquito by the introduction of a sterile insect technique is analyzed in thomé et al. [ ] . more recently, a study in disease persistence was made [ ] in brazil and otero et al. [ ] studied dengue outbreaks. all these studies were made with the aim of providing a better understanding of the nature and dynamics of dengue infection transmission. in the next section a temporal mathematical model that explores the dynamics between hosts (humans) and vectors (mosquitoes) is analyzed. the aim of this section is to present a mathematical model to study the dynamic of the dengue epidemics, in order to minimize the investments in disease's control, since financial resources are always scarce. quantitative methods are applied to the optimization of investments in the control of the epidemiologic disease, in order to obtain a maximum of benefits from a fixed amount of financial resources. the used model depends on the dynamic of the mosquito growing, but also on the efforts of public management to motivate the population to break the reproduction cycle of the mosquitoes by avoiding the accumulation of still water in open-air recipients and spraying potential zones of reproduction. the dengue epidemic model described in this paper is based on the one proposed in [ ] . it has four state variables and two control variables as follows. state variables: to describe the model it is also necessary to introduce some parameters: parameters: α r : average reproduction rate of mosquitoes α m : mortality rate of mosquitoes β : probability of contact between non-carrier mosquitoes and infected individuals η : rate of treatment of infected individuals µ : amplitude of seasonal oscillation in the reproduction rate of mosquitoes ρ : probability of individuals becoming infected θ : fear factor, reflecting the increase in the population willingness to take actions to combat the mosquitoes as a consequence of the high prevalence of the disease in the specific social environment τ : forgetting rate for goodwill of the target population ϕ : phase angle to adjust the peak season for mosquitoes ω : angular frequency of the mosquitoes proliferation cycle, corresponding to a weeks period p : population in the risk area (usually normalized to yield p = ) γ d : the instantaneous costs due to the existence of infected individuals γ s : the costs of each operation of spraying insecticides γ e : the cost associated to the instructive campaigns the model consists in minimizing subject to the following four nonlinear time-varying state equations [ ] : . . , . equation ( . a) represents the variation of the mosquitoes density per unit time to the natural cycle of reproduction and mortality (α r and α m ), due to seasonal effects µ sin(ωt + ϕ) and to human interference −x (t) and u (t). equation ( . b) expresses the variation of the mosquitoes density carrying the virus x (t) denotes the rate of the infected mosquitoes and β [x (t) − x (t)] x (t) represents the increase rate of the infected mosquitoes due to the possible contact between the uninfected mosquitoes x (t) − x (t) and infected individuals denoted by x (t). the dynamics of the infectious transmission is presented in equation ( . c). the term −ηx (t) is related to the rate of cure and ρx (t) [p − x (t)] describes the rate at which new cases spring up. the factor [p − x (t)] is the number of individuals in the area, that are not infected. equation ( . d) is a model for the level of popular motivation (or goodwill) to combat the reproductive cycle of mosquitoes. over time, the level of people motivated will have changed. as a consequence, it is necessary to invest in educational campaigns designed to increase consciousness of the population under risk. the expression −τ x (t) represents the decay of the people's motivation over time, due to forgetfulness. the term θx (t) describes the natural sensibilities of the public due to increase in the prevalence of the disease. the goal is to minimize the cost functional ( . ). this functional includes social costs related to the existence of ill individuals -like absenteeism, hospital admission, treatments -, γ d x (t), the recourses needed for the spraying of insecticide operations, γ s u (t), and for educational campaigns, γ e u (t). the model for the social cost is based on the concept of goodwill explored by nerlove and arrow [ ] . due to computational issues, the optimal control problem ( . )-( . d), that was written in the lagrange form, was converted into an equivalent mayer problem. hence, using a standard procedure (cf., section . ) to rewrite the cost functional, the state vector was augmented by an extra component x , leading to the following equivalent terminal cost problem: with given t f , subject to the control system ( . a)-( . d) and ( . ). two different implementations were considered. in a first approach, the oc problem is solved by a specific optimal control package, oc-ode, already described in section . . the oc problem considers ( . a)-( . ) and the code is available in [ ] . a second approach uses the nonlinear solver ipopt [ ] , also described in section . . in order to use this software, it was necessary to discretize the problem. the euler discretization scheme was chosen (see section . for more details). the discretization step length was h = / , because it is a good compromise between precision and efficiency. thus, the optimal control problem was discretized into the following nonlinear programming problem: the error tolerance value was − using the ipopt solver. the discretized problem, after a presolve done by the software, has variables, of which are nonlinear; and constraints, of which are nonlinear (see the ampl code for this problem in [ ] ). [ , ] that was used by the authors of the paper [ ] . it is important to salient that, at the time of the initial paper [ ] , the authors had not the same computational resources that exist nowadays. the results with oc-ode and ipopt are better since the cost to fight the dengue disease and the number of infected individuals are smaller. [ ] . packages were tested, and they could not reach a solution -some crashed at the middle or some bad scaling issues were observed. until some years ago, due to computational limitations, most of the models were run using codes made by the authors themselves as [ ] . nowadays, one can choose between several proper software packages "out of the box", that already take into account specific features of stiff problems, scaling problems, etc. with this work it is possible to realize that "old" problems can again be taken into account and be better analyzed with new technology and approaches, with the goal of finding global optimal solutions, instead of local ones. for this purpose, and at an initial research stage, it is important to understand if different kinds of discretization for an oc problem have influence on the problem resolution. this section aims to study the costs of different discretization processes, in terms of time performance, number of variables and iterations used. for the purpose of this analysis two discretization schemes, euler and second order runge-kutta's scheme [ ] (cf. section . ), are considered to solve the problem described in the previous section. this discretization process transforms the dengue epidemics problem into a standard nonlinear optimization problem, with an objective function and a set of nonlinear constraints. this nlp problem was codified, for both discretization schemes, in the ampl modeling language [ ] and can be checked in [ ] . two nonlinear solvers with distinct features were selected to solve the nlp problem: the knitro (ip method) and the snopt (sqp method). the neos server [ ] platform was used as interface with both solvers. the ipopt (ip method), used in the previous section, was the first choice for our research. however, at the time of this investigation, the neos platform was moved to another center of research and some software packages were unavailable for long periods of time. so, we had to choose another interior point robust software. table . reports the results for both solvers, for each discretization method using three different discretization steps (h = . , . , . ), rising twelve numerical experiences. the columns # var. and # const. mean the number of variables and constraints, respectively. the next columns refer to the performance measures -number of iterations and total cpu time in seconds (time for solving the problem, for evaluate the objective and the constraints functions and for input/output). as the computational experiments were made in the neos server platform, the selected machine to run the program remains unknown as well as its technical specifications. the optimal cost reached was ≈ × − for all tests. comparing the general behavior of the solvers one can conclude that the ip based method (knitro) presents much better performance than the sqp method (snopt) in terms of the measures used. regarding the knitro results, one realizes that the euler's discretization scheme has better times for h = . and h = . and similar time for h = . , when compared to runge-kutta's method. another obvious finding, for both solvers, is that the cpu time increases as far as the problem dimension increases (number of variables and constraints). with respect to the number of iterations, snopt presents more iterations as the problem dimension increases. however this conclusion cannot be taken for knitro -in fact, there does not exist a relation between the problem dimension and the number of iterations. the best version tested was knitro using runge-kutta with h = . (best cpu time and fewer iterations), and the second one was knitro with euler's method using h = . . an important evidence of this numerical experience is that it is not worth the reduction of the discretization step size because no significative advantages are obtained. at this moment, as a result of major demographic changes, rapid urbanization on a massive scale, global travel and environmental change, the world faces enormous future challenges from emerging infectious diseases. dengue illustrates well these challenges [ ] . in this work we investigated an optimal control model for dengue epidemics proposed in [ ] , that includes the mosquitoes dynamics, the effect of educational campaigns. the cost functional reflects a compromise between financial spending in insecticides and educational campaigns and the population health. for comparison reasons, the same choice of data/parameters in [ ] was considered. the results obtained from oc-ode and ipopt are similar, improving the ones previously reported in [ ] (cf. section . ). indeed, the obtained control policy in this work presents an important progress with respect to the previous best policy: the percentage of infected mosquitoes vanishes just after four weeks, while mosquitoes are completely eradicated after weeks (figures . and . ); the number of infected individuals begin to decrease after four weeks while with the previous policy this only happens after weeks (figure . ) . despite the fact that our results are better, they are accomplished with a much smaller cost with insecticides and educational campaigns (figure . ) . the general improvement, which explains why the results are so successful, rely on an effective control policy of insecticides. the proposed strategy for insecticide application seems to explain the discrepancies between the results here obtained and the best policy of [ ] . our results show that applying insecticides in the first four weeks yields a substantial reduction in the cost of fighting dengue, in terms of the functional proposed in [ ] . the main conclusion is that health authorities should pay attention to the epidemic from the very beginning: effective control decisions in the first four weeks have a decisive role in the dengue battle, and population and governments will both profit from it. we successfully solved an oc problem by direct methods using nonlinear optimization software based on ip and sqp approaches. the problem was discretized through euler and runge-kutta schemes. the implementation efforts of higher order discretization methods bring no advantages. the reduction of the discretization step and consequently the increase of the number of variables and constraints do not improve the performance with respect to the cpu time and to the number of iterations. we can point out the robustness of both solvers in spite of the dimension problem increase. the conclusions drawn in section . were helpful for decision-making future processes of discretization all over the work. as future work we intend to analyze how different parameters/weights associated to the variables in the objective function can influence the spread of the disease. this chapter was based on work available in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ ] . a model for the dengue disease transmission is presented. it consists of eight mutually-exclusive compartments representing the human and vector dynamics. it also includes a control parameter, adulticide spray, as a measure to fight the disease. the model presents three possible equilibria: two disease free equilibria (dfe) and an endemic equilibrium (ee). it has been proved that a dfe is locally asymptotically stable, whenever a certain epidemiological threshold, known as the basic reproduction number, is less than one. in this work we try to understand which is the best way to apply the control in order to effectively reduce the number of infected humans and mosquitoes. a case study, using outbreak data in in cape verde, is reported. in chapter the dengue epidemic was studied, mostly centered in people, specially in the goodwill of the individuals and spraying campaigns. however, the virus transmission scheme was overlooked: it was only considered two compartments for people and two compartments for adult mosquitoes. here, the aim is to deepen the relationship between human and mosquitoes, creating a better framework to explain the development and transmission of the disease. the mathematical model is based on [ , ] , that describes the chikungunya disease transmitted by aedes albopictus. the notation used in our mathematical model includes four epidemiological states for humans: it is assumed that the total human population (n h ) is constant, so, n h = s h + e h + i h + r h . there are also four other state variables related to the female mosquitoes (the male mosquitoes are not considered in this study because they do not bite humans and consequently they do not influence the dynamics of the disease): similarly, it is assumed that the total adult mosquito population is constant, which means n m = s m + e m + i m . in this way, we put our model more complex and reliable to the reality of dengue epidemics. for this study we introduced a control variable: the control variable, c(t), varies from to . however, the model does not fit completely the reality. epidemiologist and policy makers need to be aware of both strengths and weakness of the epidemiological modeling approach. an epidemiological model is always a simplification of reality. so, some assumptions were made to built this model: • the total human population (n h ) is constant; • there is no immigration of infected individuals into the human population; • the population is homogeneous, which means that every individual of a compartment is homogenously mixed with the other individuals; • the coefficient of transmission of the disease is fixed and does not vary seasonally; • both human and mosquitoes are assumed to be born susceptible, i.e., there is no natural protection; • for the mosquito there is no resistant phase, due to its short lifetime. to completely describe the model it is necessary to use parameters, which are: total population b : average daily biting (per day) β mh : transmission probability from i m (per bite) β hm : transmission probability from i h (per bite) /µ h : average lifespan of humans (in days) /η h : mean viremic period (in days) /µ m : average lifespan of adult mosquitoes (in days) ϕ : number of eggs at each deposit per capita (per day) µ a : natural mortality of larvae (per day) η a : maturation rate from larvae to adult (per day) /η m : extrinsic incubation period (in days) /ν h : intrinsic incubation period (in days) m : female mosquitoes per human k : number of larvae per human k : maximal capacity of larvae for notation simplicity, the independent variable t will be omitted when writing the dependent variables, example given, will be written s h instead of s h (t). the dengue epidemic can be modelled by the following nonlinear time-varying state equations: and vector population with the initial conditions for the previous set of differential equations is now analyzed the equilibrium points of the system and is determined the threshold phenomena. let the set proof. system ( . )-( . ) can be rewritten in the following way: as m (x) has all off-diagonal entries nonnegative, m (x) is a metzler matrix. using the fact that f ≥ , the system ( . ) is positively invariant in r + [ ] , which means that any trajectory of the system starting from an initial state in the positive orthant r + remains forever in r + . theorem . . . let Ω be defined as above. consider also the system ( . )-( . ) admits at most two disease free equilibrium points: • if m ≤ , there is a disease free equilibrium (dfe), called trivial equilibrium; • if m > , there is a biologically realistic disease free equilibrium (brdfe), proof. the equilibrium points are reached when the following equations hold: using the mathematica software to solve the system ( . ), we obtained four solutions. the first one is known as the trivial equilibrium, since the mosquitoes do not exist, so there is no disease: in the second one, mosquitoes and humans interact, but there is only one outbreak of the disease, i.e., over time the disease goes away without killing all the mosquitoes. we have called this equilibrium point a biologically realistic disease free equilibrium (brdfe), since it is a more reasonable situation to find in nature than the previous one: which is equivalent to e * = n h , , , kn hm η a µ b , kn hm µ b µm , , . this is biologically interesting only if m is greater than . the third solution corresponds to a situation where humans and mosquitoes live together but the disease persists in both populations, which means that it is not a dfe. this equilibrium will be explained after (see theorem . . ). thus the disease is not anymore an epidemic episode, but transforms into a endemic one. with some algebraic manipulations we obtained the following point: with the mathematica software we obtained a fourth solution. but some of the components are negative, which means that they do not belong to the Ω set. remark . the condition m > is equivalent, by algebraic manipulation, to the condition which corresponds to the basic offspring number for mosquitoes. thus, if m < , then the mosquito population will collapse and the only equilibrium for the whole system is the trivial equilibrium. if m ≥ , then the mosquito population is sustainable. the amount of mosquitoes is also related to an epidemic threshold: the basic reproduction number of the disease, r . following [ ] , we prove: . the equilibrium point brdfe is locally asymptotically stable if r < and unstable if r > . proof. to derive the basic reproduction number, we use the next generator approach. the basic reproduction number is calculated in a disease free equilibrium. in this case we consider the most realistic one, brdfe. following [ , ] , let consider the vector x t = (e h , i h , e m , i m ) which corresponds to the components related to the progression of the disease. the subsystem used is: this subsystem can be partitioned, represents the components related to new cases of disease (in this situation in the exposed compartments) and v(x) represents the other components. thus the subsystem ( . ) can be rewritten as let us consider the jacobian matrices associated with f and v: according to [ ] the basic reproduction number is r = ρ(j f (x ) j v − (x ) ), where x is a disease free equilibrium (brdfe) and ρ(a) defines the spectral radius of a matrix a. using mathematica and we obtain the value for the threshold parameter, with m > . in the model we have two different populations (human and vectors), so the expected basic reproduction number reflects the infection human-vector and also vector-human, that is, r = r hm × r mh . the term bβ hm sm n h represents the product between the transmission probability of the disease from humans to vectors and the number of susceptible mosquitoes per human; η h +µ h is related to the human's viremic period; and ηm c+ηm+µm represents the proportion of mosquitoes that survive to the incubation period. analogously, the term bβ mh s h n h is related to the transmission probability of the disease between mosquitos and human, in a susceptible population; when r < , each infected individual produces, on average, less than one new infected individual, and therefore it is predictable that the infection will be cleared from the population. if r > , the disease is able to invade the susceptible population [ , ] . theorem . . . if m > and r > , then the system ( . )-( . ) also admits an endemic equilibrium (ee): . proof. see proof of theorem . . . from a biological point of view, it is desirable that humans and mosquitoes coexist without the disease reaching a level of endemicity. good estimates of dengue transmission intensity are therefore necessary to compare and interpret dengue interventions conducted in different places and times and to evaluate options for dengue control. the basic reproduction number has played a central role in the epidemiological theory for dengue and other infectious diseases because it provides an index of transmission intensity and establishes a threshold criteria. we claim that proper use of control c, can result in the basic reproduction number remaining below unity and, therefore, making brdfe stable. in order to make effective use of achievable insecticide control, and simultaneously to explain more easily to the competent authorities its effectiveness, we assume that c is constant. the goal is to find c such that r < . for this purpose we have studied the reality of cape verde. an unprecedented outbreak was detected in the cape verde archipelago in september . this is the first report of dengue virus activity in that country. as the population had never had contact with the virus, the herd immunity was very low. dengue type spread throughout the islands of the archipelago, reaching four of the nine islands. the worst outbreak occurred on the santiago island, where most people live. the number of cases increased sharply since the beginning of november, reaching cases per day. the cape verde ministry of health reported more than cases of dengue fever within the archipelago between october and december , which is about % of the total population of the country. from cases of dengue hemorrhagic fever reported, six people died [ , ] . it represented a challenge to the performance of the national health care system. government officials launched a plan to eradicate the mosquito including a national holiday during which citizens were being asked to clear out standing water and other potential breeding areas used by the mosquitoes. the intense and spontaneous movement of solidarity from the civil society was another noteworthy dimension, not only cleaning but also voluntary donating blood to strengthen the stock of the central hospital dr. agostinho neto, in praia. we used the data for human population related to cape verde [ ] . due to low surveillance and the fact of being the first ever of dengue outbreak in the country, it was not possible to collect detailed data from the mosquito. however, the authorities speak explicitly about mosquitoes coming from brazil [ ] . also the information that comes from the ministry of health in the capital of cape verde, praia, confirms that the insects responsible for dengue came most probably from brazil, transported by means of air transport that perform frequent connections between cape verde and brazil, as reported by the radio of cape verde. with respect to aedes aegypti, we have thus considered data from brazil [ , ] . the simulations were carried out using the following values: n h = , b = , β mh = . , β hm = . , µ h = /( × ), η h = / , µ m = / , ϕ = , µ a = / , η a = . , η m = / , ν h = / , , m = , k = . the initial conditions for the problem were: considering nonexistence of control, i.e. c = , the basic reproduction number for this outbreak in cape verde is approximately r = . , which is in agreement to other studies of dengue in other countries [ ] . the control c affects the basic reproduction number, and our aim is to find a control that puts r less than one. this value was obtained by mathematica, calculating the inequality with the parameters values above. the computational investigations were carried out using c = . , which means that the insecticide is continuously applied during a twelve week period. the software used was scilab [ ] . it is an open source, cross-platform numerical computational package and a high-level, numerically oriented programming language. for our problem we used the routine ode to solve the set of differential equations. by default, ode uses the lsoda solver of package odepack. it automatically selects between the nonstiff predictor-corrector adams method and the stiff backward differentiation formula (bdf) method. it uses the nonstiff method initially and dynamically monitors data in order to decide which method to use. the graphics were also obtained using this software, with the command plot (see code in [ ] ). figures . and . show the curves related to human population, with and without control, respectively. the number of infected people, even with small control, is much less than without any insecticide campaign. figures . and . show the difference of the mosquito population with control and without control. when the control is applied, the number of infected mosquitoes is close to zero. note that the intention is not to completely eradicate the mosquitoes but instead the number of infected mosquitoes. it has been algebraically proved that if a constant minimum level of a control is applied (c = . ), it is possible to maintain the basic reproduction number below unity, guaranteeing the brdfe. this value is corroborated in another numerical study [ ] . the values of infected humans obtained by the model are higher when compared to what really happened in cape verde. despite the measures taken by the government and health authorities were not accounted, they have had a considerable impact in the progress of the disease. until here, it was considered a constant control. using a theoretical approach [ ] , we intend to find the best function c(t), using oc theory. instead of finding a constant control it will be possible to study other types of control, such as piecewise constant or even continuous but not constant. additionally we could consider another strategy, a more practical one: due to logistics and health reasons, it may be more convenient to apply insecticide periodically and at some specific hours at night. in this section we investigate the best way to apply the control in order to effectively reduce the number of infected humans and mosquitoes, using pulse control. in literature it has been proven that a dfe is locally asymptotically stable, whenever a certain epidemiological threshold, known as the basic reproduction number , is less than one. in the previous section, it was proven that if a constant minimum level of insecticide is applied (c = . ), it is possible to maintain the basic reproduction number below unity, guaranteeing the dfe. in this section, other kinds of piecewise controls that maintain the basic reproduction number less than one and could give a better solution to implement by health authorities are investigated. to solve the system ( . )-( . ), in a first step, several strategies of control application were used: three different frequencies (weekly, bi-weekly and monthly) for control application, a constant control (c = . ) and no control (c = ). the first three different frequencies mean that during one day (per week, bi-week and month), the whole ( %) capacity of insecticide (c = ) is used during all day. besides, also was used a constant control strategy (c = . ) that consists in the application of . % capacity of insecticide hours per day all the time ( days). in this work, the amount of insecticide is an adimensional value and must be considered in relative terms. the numerical tests were carried out using scilab [ ] , with the same ode system and parameters of the previous section (code available in [ ] ). figures . and . show the results of these strategies regarding infected mosquitoes and individuals. without control, the number of infected mosquitoes and individuals increase expressively. it is also possible to see that the weekly pulse control had the closest results to the continuous control. therefore, realizing the influence of the insecticide control, further tests were carried out to find the optimum periodicity of administration which, from gathered results, must rest between six and seven days. the second phase of numerical tests, figures . and . , considered four situations: days, days, days and continuously c = . . to guarantee the dfe, the curves must remain below the one corresponding to c = . . the amount of insecticide, and when to apply it, are important factors for outbreak control. table . reports the total amount of insecticide used in each version during the days. the numerical tests conclude that the best strategy for the infected reduction is every / days application. the value spent in insecticide is similar to the continuous strategy, but is much easier to implement in this work, several piecewise strategies were studied to find the best way of applying insecticide, but always having in mind a periodic application of the product. but if the best strategy is not a periodic one? in the next section the optimal control solution for this problem will be studied. the aim of this section is the study of optimal strategies for applying insecticide, taking into account different perspectives: thinking only on the insecticide cost, focusing on the cost of infected humans or even combining both perspectives. to take this approach, and after several numerical experiences, we considered that it was better to normalize the ode system ( . )-( . ). the reason for this transformation is due to the bad scaling of the variables: some vary from to , and others from to , which can influence the software performance. consider the following transformations: the ode system ( . )-( . ) is transformed into: with the initial conditions let us consider the objective functional j considering the costs of infected humans and costs with insecticide: where γ d and γ s are positive constants representing the costs weights of infected individuals and spraying campaigns, respectively. using oc theory, let λ i (t), with i = , . . . , , be the co-state variables. the hamiltonian for the present oc problem is given by by the pontryagin maximum principle [ ] , the optimal control c * should be the one that minimizes, at each instant t, the hamiltonian given by ( . ), that is, h (x * (t), λ * (t), c * (t)) = min c∈[ , ] h (x * (t), λ * (t), c). in this way, the optimal control is given by it is also necessary to consider the adjoint system λ i (t) = − ∂h ∂x i , i.e., as the oc problem just has initial conditions it is necessary to find the transversality conditions, that correspond to a terminal condition in the co-state equation, replacing the optimal control c * in the state system ( . ) and in the adjoint system ( . ) is possible to solve the differential system taking into account the initial and transversality conditions. in order to solve this oc problem three approaches were tested. the first one is a direct method, dotcvp toolbox [ ] for matlab. it uses the differential system ( . ), the initial conditions ( . ) and it is necessary to transform the problem into a mayer form (see section . ). to solve the discretized oc problem, the ipopt software is chosen as an option inside the dotcvp. the functional is divided into time intervals, in this case ten intervals, with an initial value problem tolerance of − and a nlp tolerance of − . the optimal control function given by this toolbox is piecewise constant. another direct method is oc-ode [ ] . it uses the differential system ( . ), the initial conditions ( . ) and it includes procedures for numerical adjoint estimation and sensitivity analysis and the feasibility tolerance considered was − . the last method used, an indirect one, it is coded in matlab environment. it involves the backwardforward method (see section . ) and the resolution of the ode systems are made by the ode routine. the state differential system ( . ) is solved forward with initial conditions ( . ), while the adjoint system ( . ) is solved backwards using the terminal conditions ( . ). the absolute and relative tolerances were fixed in − . the three codes are available at [ ] . all the parameters are assumed equal to the previous section. for this first simulation, the values for the weights of the costs are γ d = . and γ s = . . figure . shows that, despite having distinct philosophies of resolution, the curves obtained by the three solvers are similar, which reinforces the confidence in the result. the optimal functional is . , . and . , for dotcvp, oc-ode and backward-forward, respectively. the last solver achieved better value for the functional. this is expected, once its resolution contains more information about the oc problem because the adjoint system is supplied. the study considers three situations: a, b, and c. situation a, that was previously presented, regards both perspectives in the functional (human infected and insecticide application). situation b concerns only to infected humans whereas case c only considers insecticide campaigns. table . resumes the three situations. functional values for weights case a both perspectives since the three solvers presented similar solutions, only one of them was chosen to solve these cases, that was dotcvp. figures . , . and . show the results for optimal control c, infected humans and total costs, respectively. in the medical perspective (case b), when only the costs related with ill people (absenteeism, drugs, ...) are considered, the number of infected is the lowest; however a huge quantity of insecticide is used, because it is considered cheaper. on the other hand, when people just think on the economical perspective (case c), the treatment for people is neglected. optimal control is low, but the number of infected humans is high. the total cost is higher when both perspectives are considered. epidemiological modelling has largely focused on identifying the mechanisms responsible for epidemics but has taken little account on economic constraints in analyzing control strategies. economic models have given insight into optimal control under constraints imposed by limited resources, but they frequently ignore the spatial and temporal dynamics of the disease. nowadays the combination of epidemiological and economic factors is essential. the bioeconomic approach requires an equilibrium between economic and epidemiological parameters in order to give an efficient disease control and reflecting the nature of the epidemic. for this, the study goes on implementing both perspectives, but taking into account distinct weights of the parameters associated to the variables i h and c. table . resumes these approaches. values for weights table . : different weights for the functional in case d, it is studied a situation where the lack of insecticide in a country could be a reality and as a consequence its market value is high. this could happen due to an unprecedent outbreak where the authorities were not prepared or even due to financial reasons by the fact that the government does not have financial viability for this kind of measure. in case e, once again the human perspective gains strength and, as human life and quality of life is an expensive good, it was considered that it is more expensive to treat humans then apply insecticide. the analysis from the figures from . to . is consistent with what we expected in reality. in case d, as insecticide is expensive, the function for optimal control is lower than the other perspective. as consequence, the number of infected people is higher. in case e, where the human factor is preponderant, the number of infected humans is low but expenses with insecticide are higher. curiously the total cost, in case d and e, are of the same order of magnitude, with a slightly higher cost for case e. the total cost is reported in table . . when both perspectives are considered, the total cost is higher than the single perspective. for a last analysis, a mathematical perspective was carried out: what values should the γ d and γ s have, in order to minimize the functional? let us call this perspective as case f. in this perspective, we not only want to minimize the control c, but also the parameters γ d and γ s , enforcing the equality constraint γ d + γ s = . show the comparison of this case with the first one. as expected, the total cost and the number of infected humans are the lowest ones. giving freedom to the parameters it is possible to see that the optimal control function is not periodic (as studied in the previous section), but gives a practical solution in applying insecticide. in this chapter a model based on two populations, humans and mosquitoes, with insecticide control is presented. it has shown that as time goes by, depending on several parameters, the outbreak could disappear (leading to a dfe) or could transform the disease into an endemic one (leading to an ee). this assuming that the parameters are fixed, the only variable that can influence this threshold is the control variable c, it has shown that with a steady insecticide campaign it is possible to reduce the number of infected humans and mosquitoes, and can prevent an outbreak that could transform an epidemiological episode to an endemic disease. for a steady campaign has proven that c = . it is enough to maintain the r below unit. however, this type of control is difficult to implement. a pulse insecticide campaign was studied to circumvent this difficulty. it has proven that applying insecticide every / days, is a better strategy to implement by health authorities and has the same efficacy level and financial costs. finally, the oc theory was used to find the best optimal control function for the insecticide. the optimal function varies, giving a different answer depending on the main goal to reach, thinking in economical or human centered perspective. as future work it is important to study different kinds of controls. the accelerated increase in mosquito resistance to several chemical insecticides and the damage caused by these to the environment, has resulted in the search for new control alternatives. among the alternatives available, the use of bacillus thuringiensis israelensis (bti) has been adopted by several countries [ ] . laboratory testing shows that bti has a high larvicide property and its mechanism of action is based on the production of an endotoxin protein that, when ingested by the larvae, causes death. to ensure the minimization of the outbreaks, educational programmes that are customized for different levels of health care and that reflect local capacity should be supported and implemented widely. people should be instructed to minimize the number of potential places for the mosquito breeding. educational campaigns can be included as an extra control parameter in the model. this chapter was based on work available in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ , ] . a new model with six mutually-exclusive compartments related to dengue disease is presented. in this model there are three vector control tools: insecticides (larvicide and adulticide) and mechanical control. the human data for the model is again related to cape verde. due to the rapid development of the outbreak on the islands, only a few control measures were taken, but not quantified. in this chapter, some of these measures are simulated and their consequences are analyzed. in chapter , a model with eight compartments and a single control was analyzed. however, after discussion with some researchers in this area, many of them suggested removing the compartment exposed for three main reasons: first, it is difficult to collect data for this compartment, since the disease at this stage does not show symptoms; second, the curve obtained is similar to the infected compartment with only an advance of time, not bringing novelty to the model, but possible difficulties to the numeric resolution; and finally, as the main goal is to study the effects of several controls centered in the infected humans, this compartment plays a secondary role. thus, it was decided to remove the exposed compartments, in humans and mosquitoes, adjusting the other parameters to this new model and including three controls. taking into account the model presented in [ , ] and the considerations of [ ] - [ ] , a new model more adapted to the dengue reality is proposed. the notation used in the mathematical model includes three epidemiological states for humans: it is assumed that the total human population (n h ) is constant and n h = s h (t) + i h (t) + r h (t) at any time t. the population is homogeneous, which means that every individual of a compartment is homogeneously mixed with the other individuals. immigration and emigration are not considered. there are three other state variables, related to the female mosquitoes: due to the short lifespan of mosquitoes, there is no resistant phase. it is assumed homogeneity between host and vector populations, which means that each vector has an equal probability to bite any host. humans and mosquitoes are assumed to be born susceptible. to analyze the effect of campaigns in the disease fight, three controls are considered: proportion of mechanical control, < α ≤ . larval control targets the immature mosquitoes living in water before they become biting adults. a soil bacterium, bacillus thuringiensis israelensis (bti), is applied from the ground or by air to larval habitats. this bacterium is used because when properly applied, it has virtually no effect on non-target organisms. the control of adult mosquitoes is necessary when mosquito populations cannot be treated in their larval stage. it is the most effective way to eliminate adult female mosquitoes that are infected with human pathogens. depending on the size of the area to be treated, either trucks for ground adulticide treatments or aircraft for aerial adulticide treatments can be used. the purpose of mechanical control is to reduce the number of larval habitat areas available to mosquitoes. the mosquitoes are most easily controlled by treating, cleaning and/or emptying containers that hold water, since the eggs of the specie are laid in water-holding containers. the aim is to simulate different realities in order to find the best policy to decrease the number of infected humans. a temporal mathematical model is introduced, with mutually-exclusive compartments, to study the outbreak occurred on cape verde islands in and improving the model described in [ ] . the model uses the following parameters: average daily biting (per day) β mh : transmission probability from i m (per bite) β hm : transmission probability from i h (per bite) /µ h : average lifespan of humans (in days) /η h : mean viremic period (in days) /µ m : average lifespan of adult mosquitoes (in days) ϕ : number of eggs at each deposit per capita (per day) /µ a : natural mortality of larvae (per day) η a : maturation rate from larvae to adult (per day) m : female mosquitoes per human k : number of larvae per human the dengue epidemic is modelled by the following nonlinear time-varying state equations: with the initial conditions due to biological reasons, only nonnegative solutions of the differential system are acceptable. more precisely, it is necessary to study the solution properties of the system ( . )-( . ) in the closed set it can be verified that Ω is a positively invariant set with respect to ( . )-( . ). the proof of this statement is similar as in [ ] . the system ( . )-( . ) has at most three biologically meaningful equilibrium points (cf. theorem . . ). is said to be an equilibrium point for system ( . )-( . ) if it satisfies the following relations: an equilibrium point e is biologically meaningful if and only if e ∈ Ω. the biologically meaningful equilibrium points are said to be disease free or endemic depending on i h and i m : if there is no disease for both populations of humans and mosquitoes (i h = i m = ), then the equilibrium point is said to be a disease free equilibrium (dfe); otherwise, if i h = or i m = (in other words, if i h > or i m > ), then the equilibrium point is called endemic. theorem . . . system ( . )-( . ) admits at most three biologically meaningful equilibrium points; at most two dfe points and at most one endemic equilibrium point. more precisely, let , . if m ≤ , then there is only one biologically meaningful equilibrium point, e * , which is a dfe point. if m > with ξ ≥ χ, then there are two biologically meaningful equilibrium points, e * and e * , both dfe points. if m > with ξ < χ, then there are three biologically meaningful equilibrium points, e * , e * , and e * , where e * and e * are dfes and e * endemic. proof. system ( . ) has four solutions easily obtained with a computer algebra system like maple: e * , e * , e * and e * . the equilibrium point e * is always a dfe because it always belongs to Ω with i h = i m = . in contrast, e * is never biologically realistic because it has always some negative coordinates. the other two equilibrium points, e * and e * , are biologically realistic only for certain values of the parameters. the equilibrium e * is biologically realistic if and only if m ≥ , in which case it is a dfe. for the condition m ≤ , the third equilibrium e * is not biologically realistic. if m > , then three situations can occur with respect to e * : if ξ = χ, then e * degenerates into e * , which means that e * is the dfe e * ; if ξ > χ, then e * is not biologically realistic; otherwise, one has e * ∈ Ω with i h = and i m = , which means that e * is an endemic equilibrium point. by algebraic manipulation, m > is equivalent to condition is related to the basic offspring number for mosquitos. thus, if m ≤ , then the mosquito population will collapse and the only equilibrium for the whole system is the trivial dfe e * . if m > , then the mosquito population is sustainable. from a biological standpoint, the equilibrium e * is more plausible, because the mosquito is in its habitat, but without the disease. an important measure of transmissibility of the disease is now introduced: the basic reproduction number. it provides an invasion criterion for the initial spread of the virus in a susceptible population. for this case the following result holds. theorem . . . the basic reproduction number r associated to the differential system ( . )-( . ) is proof. in agreement with [ ] , just the epidemiological compartments that have new infections, i h and i m , are considered. the two differential equations related to these two compartments can be rewritten where f is the rate of production of new infections and v is the transition rates between states: the jacobian derivatives are the quantity j f (x) j − v(x) gives the total production of new infections over the course of an infection. the largest eigenvalue gives the fastest growth of the infected population, which means that r is the spectral radius of the matrix j f (x) j − v(x) in a dfe point. maple was used to obtain the basic reproduction number r in ( . ) is obtained, replacing s h df e and s m df e in ( . ) by those of the dfe e * . the model has two different populations (host and vector) and the expected basic reproduction number should reflect the infection transmitted from host to vector and vice-versa. accordingly, r can be seen as r = (r hm × r mh ) if r < , then, on average, an infected individual produces less than one new infected individual over the course of its infectious period, and the disease cannot grow. conversely, if r > , then each individual infects more than one person, and the disease can invade the population. proof. the only solution of ( . ) with i h > or i m > , the only endemic equilibrium, is e * . that occurs, in agreement with theorem . . , in the case m > and χ > ξ. the condition χ > ξ is equivalent, by theorem . . , to r > . using the methods in [ , ] , it is possible to prove that if r ≤ , then the dfe is globally asymptotically stable in Ω, and thus the vector-borne disease always dies out; if r > , then the unique endemic equilibrium is globally asymptotically stable in Ω, so that the disease, if initially present, will persist at the unique endemic equilibrium level. assuming that parameters are fixed, the threshold r is influenceable by the control values. figure . gives this relationship. it is possible to realize that the control c m is the one that most influences the basic reproduction number to stay below unit. besides, the control in the aquatic phase alone is not enough to maintain r below unit: an application close to % is required. the simulations were carried out using the following numerical values: n h = , b = . , β mh = . , β hm = . , µ h = /( × ), η h = / , µ m = / , ϕ = , µ a = / , η a = . , m = , k = . the initial conditions for the problem were: s h = n h − , i h = , r h = , a m = kn h , s m = mn h , i m = . with these values, one has m > . as in the previous chapter the values related to humans describe the reality of cape verde [ ] and the information about mosquitoes is based on brazil [ , ] . all computational calculus consider one year for time interval. although the final time was t f = days, the figures show graphics in suitable windows, in order to provide a better analysis. all the simulations and graphics were done in matlab. to solve the differential equation system, the ode routine was used. this function implements a runge-kutta method with a variable time step for efficient computation (see [ ] for more details about the code). the number of infected humans from the model ( . )-( . ) is higher when compared with what really happened in cape verde. as far as it was possible to investigate in the local news, the government of cape verde has done their best to banish the mosquito, with media campaigns appealing to people to remove or cover all recipients that could serve to breed the mosquito, and to use insecticide in critical areas. however, it was not possible to quantify those efforts in precise terms. next, follows a set of simulations using different controls. in each figure, only one control is used, continuously, which means that the others are not applied. the aim of this simulation is to see the importance of the control and what repercussions has on the model. figures . and . concern the adulticide control, figures . and . the larvicide control, and figures . and . the mechanical control. using a small quantity of each control, the number of infected people falls dramatically. in some cases, in spite of all graphs displaying five simulations, some curves are so close to zero that it is difficult to distinguish them. figures . and . show that excellent results for the human population are obtained by covering only % of the country with insecticide for adult mosquitoes. the simulations were done considering that the aedes aegypti does not become resistant to the insecticide and that it is financially possible to apply insecticide during all time. figures . - . are related to the applied controls in the aquatic phase of the mosquito. in these graphics the controls were studied separately, but one is closely related to the other. the application of these controls are not sufficient to decrease the infected human to zero, but the removal in the next section, using oc strategy we will find the best solution for the controls. epidemiological models may give some basic guidelines for public health practitioners, comparing the effectiveness of different potential management strategies. in reality, a range of constraints and trade-offs may substantially influence the choice of practical strategy, and therefore their inclusion in any modelling analysis may be important. frequently, epidemiological models need to be coupled to economic considerations, such that control strategies can be judged through holistic cost-benefit analysis. control of livestock disease is a scenario when cost-benefit analysis can play a vital role in choosing between cheap, weak controls that lead to a prolonged epidemic, or expensive but more effective controls that lead to a shorter outbreak. normalizing the previous ode system ( . )-( . ), we obtain: with the initial conditions the cost functional considered was where γ d , γ s , γ l and γ e are weights related to costs of the disease, adulticide, larvicide and mechanical control, respectively. in a first approach to this problem, it is assumed that all weights are the same, which means γ d = γ s = γ l = γ e = . (case a). the oc problem was solved using two different packages: dotcvp [ ] and muscod-ii [ ] . the mathematical formulation of the sir+asi problem, for the both packages, is available on [ ] . the simulation behavior is similar, and we decided to show only the dotcvp results. the optimal functions for the controls are given in figure . . the adulticide was the control that more influenced the decreasing of that ratio and, as consequence, the decreasing of the number of infected people and mosquitoes, matching with the results obtained for the basic reproduction number in section . . therefore, the adulticide was the most used. we believe that the other controls do not assume an important role in the epidemic episode, because all the events happened at a short period of time, which means that adulticide has more impact. however the mosquito control in the aquatic phase can not be neglected. in situations of longer epidemic episodes or even in an endemic situation, the larval control represents an important tool. figure . presents the number of infected humans. comparing the optimal control case with a situation with no control, the number of infected people decreased considerably. besides, in the situation where oc is used, the peak of infected people is minor, which facilitates the work in health centers, because they can provide a better medical monitoring. a second analysis was made, taking into account different weights on the functional. table . resumes the weights chosen for perspectives: not only economic issues (cost of insecticides and educational campaigns), but also human issues are considered. in case a, all costs were equal. in case b is given more impact on the infected people, considering that the treatment and absenteeism to work is very prejudicial to figure . : comparison of infected individuals under an optimal control situation and no controls the country, when compared with the cost of insecticides and educational campaigns. in case c, the costs with killing mosquitoes and educational campaigns have more impact in the economy. higher total costs were obtained when human life had more weight than controls measures as can be checked in table table . : different weights for the functional and respective values figure . shows the number of infected human in each bioeconomic perspective. we can realize that case a and case c are similar. it can be explained by the low weight given to the cost of treatment (cases a and c) when compared with the heavy weight given in case b. figure . presents the behavior of the controls for the a, b and c cases. again, as adulticide is the one that has more influence on the model, this is the control that most varies when the weights are changed. a third analysis was made to the model: it changed the functional in order to study the effects of each control when separately considered. therefore, the new functional also considers bioeconomic perspectives, but only includes two variables: the costs with infected humans (with γ d = . ) and the costs with only one control (with γ i = . , i ∈ {s, l, e}). thus, in figure . are presented the proportion of adulticide (a) and infected humans (b), when the functional is dengue disease breeds, even in the absence of fatal forms, significant economic and social costs: absenteeism, debilitation and medication. to observe and to act at the epidemics onset could save lives and resources to governments. moreover, the under-reporting of dengue cases is probably the most important barrier in obtaining an accurate assessment. a compartmental epidemiological model for dengue disease composed by a set of differential equations was presented. simulations based on clean-up campaigns to remove the vector breeding sites, and also simulations on the application of insecticides (larvicide and adulticide), were made. it was shown that even with a low, although continuous, index of control over time, the results are surprisingly positive. the adulticide was the most effective control, since with a low percentage of insecticide, the basic reproduction number is kept below unit and the number infected humans was smaller. however, to bet only in adulticide is a risky decision. in some countries, such as mexico and brazil, the prolonged use of adulticides has been increasing the mosquito tolerance capacity to the product or even they become completely resistant. in countries where dengue is a permanent threat, governments must act with differentiated tools. it will be interesting to analyze these controls in an endemic region and with several outbreaks. we believe that the results will be quite different. aedes aegypti eradication is not considered to be feasible and, from the environmental point of view, not desirable. the aim is to reduce the mosquito density and, simultaneously, amount the level of immunity on the human population. the increase of population herd immunity can be reached by two ways: increasing the resistant people to the disease implying an increasing of infected individuals or with a vaccination campaign. no commercially available clinical cure or vaccine is currently available for dengue, but efforts are underway to develop one [ , ] . the potential of prevention of dengue by immunization seems to be technically feasible and progress is being made in the development of vaccines that may protect against all four dengue viruses. in the next chapter, a model of this disease with a vaccine simulation as a new strategy to fight the disease will be analyzed. this chapter was based on work accepted in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ ] . as the development of a dengue vaccine is ongoing, it is simulated an hypothetical vaccine as an extra protection to the population. in a first phase, the vaccination process is studied as a new compartment in the model, and some different types of vaccines are simulated: pediatric and random mass vaccines, with distinct levels of efficacy and durability. in a second step, the vaccination is seen as a control variable in the epidemiological process. in both cases, epidemic and endemic scenarios are included in order to analyze distinct outbreak realities. in , the swiss mathematician daniel bernoulli published a study on the impact of immunization with cowpox upon the expectation of life of the immunized population [ ] . the process of protecting individuals from infection by immunization has become a routine, with historical success in reducing both mortality and morbidity. the impact of vaccination may be regarded not only as an individual protective measure, but also as a collective one. while direct individual protection is the major focus of a mass vaccination program, the effects on population also contribute indirectly to other individual protection through herd immunity, providing protection for unprotected individuals [ ] (see scheme in figure . ). this means that when we have a large neighborhood of vaccinated people, a susceptible individual has a lower probability in coming into contact with the infection, being more difficult for diseases to spread, which decreases the relief of health facilities and can break the chain of infection. vector control remains the only available strategy against dengue. despite integrated vector control with community participation, along with active disease surveillance and insecticides, there are only a [ ] . besides, the levels of resistance of aedes aegypti to insecticides has increased, which implies shorter intervals between treatments, and only few insecticide products are available in the market due to the high costs for development and registration and low returns [ ] . dengue vaccines have been under development since the s, but due to the limited appreciation of global disease burden and the potential markets for dengue vaccines, industry interest languished throughout the th century. however, in recent years, the development of dengue vaccines has accelerated dramatically with the increase in dengue infections, as well as the prevalence of all four circulating serotypes. faster development of a vaccine became a serious concern [ ] . economic analysis are now conducted periodically to guide public support for vaccine development in both industrialized and developing countries, including a previous cost-effectiveness study of dengue [ , , ] . the authors compared the cost of the disease burden with the possibility of making a vaccination campaign; they suggest that there is a potential economic benefit associated with promising dengue interventions, such as dengue vaccines and vector control innovations, when compared to the cost associated to the disease treatments. constructing a successful vaccine for dengue has been challenging: the knowledge of disease pathogenesis is insufficient and in addition the vaccine must protect simultaneously against all serotypes in order to not increase the level of dhf [ ] . nevertheless, several promising approaches are being investigated in both academic and industrial laboratories. vaccine candidates include live attenuated vaccines obtained via cell passages or by recombinant dna technology (such as those being developed by the us national institutes of allergy and infectious diseases, inviragen, walter reed army institute of research/glaxosmithkline, and sanofi pasteur) and subunit vaccines (such as those developed by merck/hawaii biotech) [ , ] . recent studies indicate that, by the progress in clinical development of sanofi pasteur's live attenuated tetravalent chimeric vaccine, a vaccine could be licensed as early as [ ] . the team is carrying out an efficacy study on a vaccine covering four serotypes on children aged four to eleven years old in muang district, thailand. at this time, the features of dengue vaccine are mostly unknown. so, in this chapter we opt to present a set of simulations with different types of vaccines and we have explored also the vaccination process under two different perspectives. the first one uses a new compartment in the model and several kinds of vaccination are considered. a second perspective is studied using the vaccination process as a disease control in the mathematical formulation. in this case the theory of oc is applied. both methods assume a continuous strategy vaccination. in this section, a new compartment v is added to the previous sir model related to the human population. this new compartment represents the new group of human population that is vaccinated, in order to distinguish the resistance obtained through vaccination and the one achieved by disease recovery. two forms of random vaccination are possible: the most common for human disease is pediatric vaccination to reduce the prevalence of an endemic disease; the alternative is random vaccination of the entire population in an outbreak situation. in both types, the vaccination can be considered perfect conferring % protection for all life or else imperfect. this last case can be due to the difficulty of producing an effective vaccine, the heterogeneity of the population or even the life span of the vaccine. for many potentially human infections, such as measles, mumps, rubella, whooping cough, polio, there has been much focus on vaccinating newborns or very young infants. dengue can be a serious candidate for this type of vaccination. in the sv ir model, a continuous vaccination strategy is considered, where a proportion of the newborn p (where ≤ p ≤ ), was by default vaccinated. this model also assumes that the permanent immunity acquired through vaccination is the same as the natural immunity obtained from infected individuals eliminating the disease naturally. the population remains constant, i.e., n h = s h + v h + i h + r h . the new model for human population is represented in figure we are assuming that it is a perfect vaccine, which means that it confers life-long protection. as a first step, it is necessary to determine the basic reproduction number without vaccination (p = ). theorem . . . the basic reproduction number, r , associated to the differential system ( . ) without vaccination is given by proof. the proof of this theorem is similar to the one in the previous chapter (see proof of theorem . . ). just consider in this chapter, we make all the simulations in two scenarios: an epidemic and an endemic situation (programming codes available in [ ] ). for these, the following parameter values of the differential system and initial conditions were used (tables . and . ). there were two main differences between the epidemic episode and an endemic situation. firsty, in the endemic situation there was a slight decrease in the average daily biting b and transmission probabilities β mh and β hm , that could be explained by the fact that the mosquito could have more difficulties to find a naive individual. the second difference is concerned with the strong increase of the initial human population that is resistant to the disease. this may be explained by the fact that the disease, in an endemic situation, already creates an immune resistance to the infection, i.e., the population already has herd immunity. with these values we obtain approximately r = . and r = . for epidemic and endemic scenarios, respectively. during an outbreak, the disease transmission assumes different behaviors, according to the distinct scenarios, as can been seen in figure . . in one year, the peak in an epidemic situation could reach more than cases. in contrast, instead in the endemic situation the curve of infected individuals has a more smooth behavior and reaches a peak less than cases. figure . relates to the mosquito population. in the endemic scenario, because a substantial part of the human population is resistant to the disease, the infected mosquitoes bite a considerable percentage of resistant host, and as consequence, the disease is not transmitted. suppose that at time t = a proportion p of newborns are vaccinated with a perfect vaccine that causes no side effects. since this proportion, p, is now immune, r is reduced, creating a new basic reproduction number. definition (basic reproduction number with pediatric vaccination). the basic reproduction number with pediatric vaccination, r p , associated to the differential system ( . ) is given by where r is defined in ( . ). observe that r p ≤ r . equality is only achieved when p = , i.e., when there is no vaccination. the constraint r p < implicitly defines a critical vaccination portion p > p c that must be achieved for eradication, where since vaccination entails costs, to choose the smallest coverage that achieves eradication is the best option. this way, the entire population does not need to be vaccinated in order to eradicate the disease. this phenomenon is called herd immunity. vaccinating at the critical level, p c , does not instantly lead to disease eradication. the immunity level within the population requires time to build up and at the critical level it may take a few generations before the required herd immunity is achieved. thus, from a public health perspective, p c acts as a lower bound on what should be achieved, with higher levels of vaccination leading to a more rapid elimination of the disease. figure . shows the simulations related to the proportion of newborns vaccinated (p = , . , . , . , ) in both scenarios. notice that at time t = no person was vaccinated. in the epidemic situation, as the outbreak reached a peak at the beginning of the year, the proportion of newborns vaccinated at that time is minimum and cannot influence the curve of infected individuals, giving the optical illusion of a single curve. on the other hand, in the endemic case, as the outbreak occurs later, the vaccination campaign starts to produce effects, decreasing the total number of sick humans. this last graphic illustrates that a vaccination campaign centered in newborns is a bet for the future of a country, but does not produce instantly results to fight the disease. to produce immediate results, it is necessary to use random mass vaccination, which means that it is necessary to vaccine a significant part of the population. a mass vaccination program may be initiated whenever there is an increase of the risk of an epidemic. in such situations, there is a competition between the exponential increase of the epidemic and the logistical constraints upon mass vaccination. for most human diseases it is possible, and more efficient, to not vaccinate those individuals who have recovered from the disease because they are already protected. another situation could be the introduction of a new vaccine in a population that lives an endemic situation. let us consider the control technique of constant vaccination of susceptibles. in this scheme a fraction ≤ ψ ≤ of the entire susceptible population, not just newborns, is being continuously vaccinated. it is assumed that the permanent immunity acquired by vaccination is the same as natural immunity obtained from infected individuals in recovery. the epidemiological scheme is presented in figure the mathematical formulation for human population (the differential equations related to the mosquito remain equal to the previous subsection) is given by: for this model, we define a new basic reproduction number. definition (basic reproduction number with random mass vaccination [ ] ). the basic reproduction number with random mass vaccination, r ψ , associated to the differential system ( . ) is where r is defined in ( . ) . comparing this model with the constant vaccination of newborns model, it is apparent that instead of constantly vaccinating a portion of newborns, a part of the entire susceptible population is now being continuously vaccinated. since the natural birth rate µ h is usually small, the fraction pµ h of newborns being continuously vaccinated will be small, whereas in this model, will be also a larger group of susceptibles can be continuously vaccinated, ψs h . due to this, we expect that this model should require a smaller proportion of ψ to achieve eradication. notice that r ψ ≤ r . equality is only achieved in the limit ψ = , that is, when there is no vaccination. the constraint r ψ < implicitly defines a critical vaccination portion ψ > ψ c that must be achieved for eradication, where ψ c = (r − ) µ h . observe that in spite of the calculations done in the period of days, the figures only show suitable windows, in order to provide a better analysis. in both scenarios, even with a small coverage of population, vaccination dramatically decreases the number of infected. the epidemic scenario has changed from less than cases (with no vaccination, figure . ) to less than cases vaccinating only % of population. in the endemic scenario, the decrease is more accentuated. until here, we have considered a perfect vaccine, which means that every vaccinated individual remains resistant to the disease. however, a majority of the available vaccines for the human population does not produce % success in the disease battle. usually, the vaccines are imperfect, which means that a minor percentage of cases, in spite of vaccination, are infected. most of the theory on the disease evolution is based on the assumption that the host population is homogeneous. individual hosts, however, may differ and they may constitute very different habitats. in particular, some habitats may provide more resources or be more vulnerable to virus exploitation [ ] . the use of models with imperfect vaccines can describe better this type of human heterogeneity. another explanation for the use of imperfect vaccines is that until now we had considered models that assumed that as soon as individuals begin the vaccination process, they become immediately immune to the disease. however, the time it takes for individuals to obtain immunity by completing a vaccination process cannot be ignored, because meanwhile an individual can be infected. in this section a continuous vaccination strategy is considered, where a fraction ψ of the susceptible class was vaccinated. the vaccination may reduce but not completely eliminate susceptibility to infection. for this reason we consider a factor σ as the infection rate of vaccinated members. when σ = the vaccine is perfectly effective and when σ = the vaccine has no effect at all. the value − σ can be understood as the efficacy level of the vaccine. the new model for the human population is represented in figure . . figure . : epidemiological sv ir model for human population with an imperfect vaccine therefore, the differential system is as follows: ( . ) for this system of differential equations, we have a new basic reproduction number. [ ] ). the basic reproduction number with an imperfect vaccine, r σ , associated to the differential system ( . ) is where r is defined in ( . ) . notice that r ψ ≤ r σ and when the vaccine is perfect (σ = ), r σ degenerates into r ψ . in other words, a high efficacy vaccine leads to a lower vaccination coverage to eradicate the disease. however, it is noted in [ ] that it is much more difficult to increase the efficacy level of the vaccine when compared to controlling the vaccination rate ψ. figure . a, in the epidemic scenario with a perfect vaccine, the number of human infected has reached to a maximum peak of cases per day, in the worst scenario (ψ = . ). using an imperfect vaccine, with a level of efficacy of % (figure . a), with the same values for ψ, the maximum peak increases until cases. we conclude that production of a vaccine with a high level of efficacy has a preponderant role in the reduction of the disease spread. figures . c and . d reinforce the previous sentence. assuming that % of the population is vaccinated, the numbers of infected cases decreases sharply with the increasing of the effectiveness level of the vaccine. according to [ ] , an acceptable level of efficacy is at least % against all four serotypes, and to years for the length of protection. these are commonly considered across countries, as the minimum acceptable levels. in the next subsection we study another type of imperfect vaccine: a vaccine that confers a limited life-long protection. until the s, this was an universal assumption of mathematical models of vaccination: there is no waning of vaccine-induced immunity. this assumption was routinely made because, for most of the major vaccines against childhood infectious diseases, it is approximately correct [ ] . suppose that the immunity, obtained by the vaccination process, is temporary. assume that immunity has the waning rate θ. then the model for humans is given by definition (basic reproduction number with an imperfect vaccine). the basic reproduction number with an imperfect vaccine, r θ , associated to the differential system ( . ) is where r ψ is defined in ( . ). according to [ ] , the basic reproduction numbers are the same, r θ and r ψ , because the disease will still spread at the same rate with or without temporary immunity. however, we should expect that the convergence rate will be different between the random mass vaccination and random mass vaccination with waning immunity, since the disease will be eradicated faster in the constant treatment model without waning immunity compared to the other with waning immunity. depending on the vaccine that will be available on the market, it will be possible to choose or even combine features. in the next section we will define the vaccination process as a control system. in this section we consider a sir model for humans and an asi model for mosquitoes. the parameters remain the same as in the previous chapter. the vaccination is seen as a control variable to reduce or even eradicate the disease. let u be the control variable related to the proportion of susceptible humans that are vaccinated. a random mass vaccination with waning immunity is selected. in this way, a parameter θ associated to the control u represents the waning immunity process. figure . shows the epidemiological scheme for the human population. figure . : epidemiological sir model for the human population using the vaccine as a control the model is described by an initial value problem with a system of six differential equations: the main aim is to study the optimal vaccination strategy, considering both the costs of treatment of infected individuals and the costs of vaccination. the objective is to where γ d and γ v are positive constants representing the weights of the costs of treatment of infected and vaccination, respectively. using oc theory is possible to solve the problem. let us consider the following set of admissible control functions: ∆ = {u(·) ∈ (l ∞ ( , t f ))| ≤ u(t) ≤ , ∀t ∈ [ , t f ]}. table ( . ), admits a unique optimal solution (s * h (·), i * h (·), r * h (·), a * m (·), s * m (·), i * m (·)) associated with an optimal control u * (·) on [ , t f ], with a fixed final time t f . moreover, there exists adjoint functions, λ * i (·), i = ... such that with the transversality conditionsλ i (t f ) = , i= , . . . . furthermore, u * = min , max , proof. the existence of optimal solutions (s * h (·), i * h (·), r * h (·), a * m (·), s * m (·), i * m (·)) associated to the optimal control u * (·) comes from the convexity of the integrand of the cost function ( . ) with respect to the control u and the lipschitz property of the state system with respect to state variables (s h , i h , r h , a m , s m , i m ) (for more details see [ ] ). according to the pontryagin maximum principle [ ] , if u * (·) ∈ ∆ is optimal for the problem considered, then there exists a nontrivial absolutely continuous mapping λ : [ , t f ] → r, λ(t) = (λ (t), λ (t), λ (t), λ (t), λ (t), λ (t)), called the adjoint vector, such that ( . ) and holds almost everywhere on [ , t f ]. moreover, the transversality conditions λ i (t f ) = , i = , . . . , hold. system ( . ) is derived from ( . ), and the optimal control ( . ) comes from the minimality condition ( . ). the simulations were carried out using the values of the previous section. the system was normalized, using the same strategy as in chapter . it was considered that the waning immunity was at a rate of method epidemic scenario endemic scenario direct (dotcvp) . . indirect (backward-forward) . . table . : optimal values of the cost functional ( . ) θ = . . the oc problem was solved using two methods: direct [ , ] and indirect [ ] . the direct method uses the cost functional ( . ) and the state system ( . ) and was solved by dotcvp [ ] . the indirect method used is an iterative method with a runge-kutta scheme, solved through ode of matlab. figure . shows the optimal control obtained by both methods. notice that dotcvp only gives the optimal control as a constant piecewise function. table . shows the costs obtained by the two methods in both scenarios. the indirect method gives a lower cost. this method uses more mathematical theory about the problem, such as the adjoint system ( . ) and optimal control expression ( . ). therefore it makes sense that the indirect method produces a better solution. using the optimal solution as reference, some tests were performed, regarding infected individuals and costs, when no control (u ≡ ) or upper control (u ≡ ) is applied. table . shows the results for dotcvp in the three situations. in both scenarios, using the optimal strategy of vaccination produces better costs with the disease, when compared to not doing anything. once there is no control, the number of infected humans is higher and produces a more expensive cost functional. figure . shows the number of infected humans when different controls are considered. it is possible to see that using the upper control, which means that everyone is vaccinated, implies that just a few individuals were infected, allowing eradication of the disease. although the optimal control, in the sense of objective ( . ), allows the occurrence of an outbreak, the number of infected individuals is much lower when compared with a situation where no one is vaccinated. we conclude that a vaccination campaign in the susceptible population, and assuming a considerable efficacy level of the vaccine, can quickly decrease the number of infected people. the worldwide expansion of the dengue fever is a growing health problem. dengue vaccine is an urgent challenge that needs to be overcome. this may be commercially available within a few years, when the researchers find a formula that protect against all four dengue viruses. a vaccination program is seen as an important measure used in infectious disease control and immunization and eradication programs. in the first part of the chapter, different types of vaccine, as well as their features and some of coverage thresholds, were introduced. the main idea was to study some types of vaccines in order to cover most of the future vaccine features. the main goal of a vaccination program is to reduce the prevalence of an infectious disease and ultimately to eradicate it. it was shown that eradication success depends on the type of vaccine as well as on the vaccination coverage. the imperfect vaccines may not completely prevent infection but could reduce the probability of being infected, thereby reducing the disease burden. in this study all the simulations were done using epidemic and endemic scenarios to illustrate distinct realities. a second analysis was made, using an oc approach. the vaccine behaved as a new disease control variable and, when available, can be a promising strategy to fight the disease. dengue is an infectious tropical disease difficult to prevent and manage. researchers agree that the development of a vaccine for dengue is a question of high priority. in the present study we have shown how a vaccine results in saving lives and at the same time in a reduction of the budget related with the disease. as future work we intend to study the interaction of a dengue vaccine with other kinds of control already investigated in the literature, such as insecticide and educational campaigns [ , ] . this chapter was based on work accepted in the peer reviewed proceedings [ ] . mathematical models can be a powerful tool to understand epidemiological phenomena. these models can be used to compare, plan, implement and evaluate several programs related to detection, prevention and control of infectious diseases. indeed, one of the most important issues in epidemiology is to improve control strategies with the final goal to reduce or even eradicate the disease. in this thesis were constructed and analyzed some models for the spreading of diseases, particularly for dengue fever. the links between health and sustainable development are illustrated for this disease. any attempt in predicting and preventing the disasters caused by the disease will imply a global strategy that takes into account environmental conditions, levels of poverty and illiteracy and, eventually, degree of coverage by vaccination programs [ ] . although vector control strategies were already available before the second world war, dengue pandemic was underestimated. it became a global public health problem in the past years and a major concern for who. the main contributions of this thesis can be classified into three main categories, namely, model formulation, mathematical and computational analysis, and contributions to public health/intervention design. deterministic models for assessing the combined impact of several control measures in dengue disease were considered. in chapter an old oc problem for dengue was revisited. it was shown that new and robust tools bring refreshing solutions to the problem. some analysis with discretization schemes were carried out in order to understand the best way to implement the direct methods in future approaches. for this problem, is was better to use robust solvers than to implement higher order discretization methods, due to the increasing of problem's dimension. in chapter , a seir+asei model was studied. threshold criteria was established ensuring the disease eradication and hence convergence to the so-called disease free solution. using real data from cape verde, the application of insecticide in the country during the outbreak was simulated and its repercussions were analyzed. the study of this outbreak was performed in several phases: firstly, using a previously calculated constant control for the whole period, with the aim of having a basic reproduction number below unity; then, several periodic strategies were studied in order to find the best logistics approaches to implement that keep r less than one; finally, an oc approach to compare with the previous suboptimal approaches was used. for this final phase, several perspectives of the problem were analyzed, including bioeconomic, medical and economic approaches. depending of the main target to achieve, the results for the control and infected individuals vary. in chapter , a sir+asi model for dengue was presented. the lost of two differential equations, was compensated by the introduction of two more controls: in addition to adulticide, larvicide and mechanical control were introduced. similarly to chapter , a threshold criteria for the eradication of the disease was established. the influence of the controls in this threshold was analyzed. then, varying the controls, separately and simultaneously, an analysis of the importance/consequence of each control in the development of the disease was made. finally, an oc approach using different weights for the variables in the functional was studied, in order to establish the best optimal curve for each control and the respective effects in the development/erradication of the disease. in chapter , some simulations with different types of vaccines were made. as a dengue vaccine is not yet available, distinct hypothetical models to introduce the vaccine were studied. in section . , was considered a new compartment v h producing a new model svir+asi. this research comprised svir models with perfect vaccines -constant vaccination of newborns and constant vaccination of susceptibles -and imperfect vaccines -using a level of efficacy below % and also with waning immunity. in section . , the vaccination process was studied as a control of the epidemic model. for this, as in the previous chapter, an oc approach was presented. all the simulations were done in epidemic and endemic scenarios, in order to understand what type of repercussions could bring each kind of vaccines. in this work, there was a concern in producing a mathematical analysis for all new models presented. epidemiological concepts, such as the basic reproduction number and equilibrium points, were calculated. the equilibrium points were classified and their local stability analyzed. the oc theory was used in order to provide the best strategies for each model, involving direct and indirect approaches. throughout the thesis, a set of software packages were used, showing the importance of the these tools in the development of some mathematical fields. to solve ode systems, codes in matlab and scilab were implemented. to calculate the equilibrium points and the basic reproduction number, mathematica and maple were used. for the oc approach, several packages were also selected: from programmed codes in ampl and run in neos server (ipopt, snopt, knitro, muscod-ii) to fortran codes in the linux environment (oc-ode) or even programs coded in matlab (dotcvp and indirect methods). the software choice was varying during the research process, due to availability, robustness and solving speed. the main codes developed in this thesis are available at: https://sites.google.com/site/hsofiarodrigues/home/ phd-codes- [ ] . using direct and indirect methods, the solutions obtained were similar, reinforcing the confidence in the results. the study provides some important epidemiological insights into the impact of vector control measures. dengue burden decreases with the increasing of vector control measures (adulticide, larvicide and mechanical control). furthermore, the adulticide should be the first/main measure to apply when an outbreak occurs, whereas the other two measures should be considered as a long time prevention. the last control measure to be studied was the vaccination. it was shown that the vaccine, when available, could bring advantages not only in the reduction of infected individuals, but also decreasing the disease costs. a phd thesis is always an unfinished process, but some day it is necessary to stop. therefore, some topics were not explored and can be understood as future directions of the work. a first suggestion is to use heterogeneity for both populations, dividing each one in more compartments [ ] . human population is not immunologically homogeneous, presenting groups with distinct level of risk, related with age, sex or the presence of a disease or immunosuppressive drugs which would be the case with transplant patients or cancer suffers. for example, the transmission probability in young children is higher and generally with more severe symptoms when compared to adult transmission. moreover, the mosquito population does not have the same behavior during the whole year, depending specially on weather conditions. temperature and humidity are key variables on vector population dynamics. it will be interesting to add some seasonality factors into the last models [ , ] . another open question is the introduction of immigration and tourism issues. along the work it was considered a constant population, but the addition of new individuals could induce new outbreaks. other aspect is related to the disease development in the presence of several serotypes. while in cape verde only one serotype was found, the interaction of several serotypes in asia is already a reality. this will induce changes in the model, not only increasing of the number of variables but also causing a more expressive number of dhf cases [ ] . currently, portuguese researchers are developing a new repellent for mosquito that could be considered as a new control for the disease. this product does not kill the mosquito, but prevents the bite by deviating it from the target and consequently decreasing the chain of the disease transmission. with the viability of the vaccine, it will be possible to fit a better model according to the vaccine used. one of the possibilities is using pulse vaccination, where children at a certain age cohorts are periodically immunized [ ] . the theoretical challenge of pulse vaccination is the a priori determination of the pulse interval for specific values of r , the proportion of vaccinated p and the population birth rate µ. most of the analysis and models presented in this thesis can be adapted for other vector-borne diseases -such as malaria, yellow fever, west nile virus, chikungunya, japanese encephalitis -by just fitting some variables/parameters and some initial assumptions intrinsic to the disease [ , , ] . when attempting to model epidemics and control for public health applications, there is the compelling urge to make models as sophisticated as possible, including many details about the host and the vector. although this strategy may be useful when such details are known or exist suitable data, it may lead to a false sense of accuracy when reliable information is not available. another approach is to keep the model simple and, instead of using the conventional differential calculus, to apply fractional calculus to fit to the disease reality [ ] or to use the general theory of time scales [ ] . 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self-control people's success at goal pursuit. however, covid- self-quarantine measures and country-level lockdowns have largely challenged people's ability to stick to their daily routines and habits. how successful at goal pursuit are people with high self-control when the world around them is not as it used to be? we examined if self-control passes the ‘quarantine test’. in an online study (n = ), we measured trait self-control, goal progress, continued engagement in pre-pandemic goal-directed behaviors, development of new goal-directed behaviors and turning these new behaviors into habits. results showed that during lockdown, people with higher (vs. lower) trait self-control were not only more likely to continue engaging in pre-pandemic goal-directed behaviors, but also found it easier to develop new goal-directed behaviors and were more likely to turn these behaviors into habits. high self-control people's ability to continue performing pre-pandemic goal-directed behaviors and to turn new behaviors into habits explained their success at goal attainment despite the major disruptions caused by the pandemic. quarantine measures implemented to fight the covid- pandemic have radically changed the lives of millions of people worldwide. besides having various negative consequences for psychological wellbeing (brooks et al., ; rajkumar, ; rodriguez, litt, & stewart, ) , quarantine measures are likely to pose major challenges to personal goal pursuit because they disrupt people's daily routines and make it hard or even impossible to continue engaging in behaviors people used to engage in to reach their goals. people's struggle to stay on track and keep pursuing their goals despite major upheavals in almost all spheres of life has been often addressed in the popular press (samuel, ; times staff, ) . what predicts individuals' ability to stay on track and stick to their goals despite major disruptions in their lives? the literature has long identified a personality trait that predicts successful goal pursuit: self-control. people with higher (vs. lower) selfcontrol are more successful at reaching their goals across various life domains, including health, well-being, relationships, academic, career, financial and others (de ridder, lensvelt-mulders, finkenauer, stok, & baumeister, ; tangney, baumeister, & boone, ) . herein, we asked whether trait self-control also passes the 'quarantine test': using the covid- pandemic as a natural laboratory to study goal pursuit under exceptionally disruptive circumstances, we examined whether trait self-control is associated with more goal progress. prior research on self-control has shown that one of the reasons why people with higher (vs. lower) self-control are more successful at goal attainment is because they use adaptive behavioral strategies, such as turning goal-directed behaviors into habits (adriaanse, kroese, gillebaart, & de ridder, ; de ridder & gillebaart, ; ent, baumeister, & tice, ; galla & duckworth, ; stavrova, pronk, & kokkoris, ; stavrova, pronk, & kokkoris, ) . this tendency to develop "beneficial habits" could facilitate goal attainment during selfquarantine for high self-control individuals. herein, we examined whether during the pandemic people with higher (vs. lower) trait selfcontrol were more likely to stick to their "beneficial habits" and continue engaging in behaviors they had developed to reach their goals prior to the pandemic. at the same time, regardless of whether high self-control people were more likely to continue engaging in their pre-pandemic goal-directed behaviors, it is intriguing whether self-control promotes flexibility and the ability to develop new goal-directed behaviors to adapt to the current situation as well. developing new adaptive strategies might be necessary for goal pursuit when external circumstances change and existing routines may no longer serve one's goals. although it has been argued that self-control might also be instrumental in the https://doi.org/ . /j.paid. . received july ; received in revised form october ; accepted october ⁎ corresponding author. t development of new habits (e.g., wood, ) , there has not been much empirical research examining the relation between self-control and new habit formation. the covid- pandemic provides a very suitable context for the study of initial formation of beneficial habits. thus, we investigated whether people with higher self-control would be more flexible to adjust to the new situation, would find it easier to develop new behaviors to reach their goals and would be more likely to turn these behaviors into habits that support their goal pursuit. in sum, we used the covid- pandemic as a context to examine whether people with higher (vs. lower) self-control made more progress towards their goals, and whether they did so by continuing performing their pre-existing goal-directed behaviors and/or by being more flexible and able to develop new behaviors and turn them into habits. participants were undergraduate students ( women, - years old, m = . , sd = . ) recruited via the subject pool of a large european university for course credit. a sensitivity power analysis showed that this sample size can reliably detect effect sizes of ρ = . (two-tailed) with an alpha level of . and power of . . the study was conducted online from june to , . after an explanation of what goals are (stavrova et al., ; stavrova et al., ) , participants were asked to write down one personal goal that they had started pursuing before the lockdown and that they still wanted to achieve. then, they wrote down up to five behaviors they had been using to achieve this goal before the lockdown started. for each behavior, they indicated to what extent they were able to continue performing it after the lockdown started (α = . ; = not at all; = very much). participants were also asked whether they had developed any new behaviors to achieve their goals after the lockdown started (yes/no), and if yes, to write down up to five of them. for each one of the new behaviors, they indicated how easy it was to develop this behavior (α = . ; = not at all; = very much) and to what extent it had already become a habit for them, i.e. part of their daily routine (α = . ; = not at all; = very much). goal progress was assessed with three items (α = . ) adopted from milyavskaya and inzlicht ( ) ; e.g., "i feel like i'm on track with my goal plan"; = strongly disagree; = strongly agree). in addition, participants filled out a measure of flexibility regarding their overall response to the covid- crisis, which we developed for the purpose of this study. it comprised the following five items (α = . ): "i accepted the challenge to reconsider my routines," "i responded to the demands of the situation with flexibility," "i tried to adjust to the new situation as best as i could," "i discovered new ways of doing things," and "i found alternative solutions to my problems" ( = not at all true; = very true). finally, trait self-control was measured with the brief self-control scale (tangney et al., ) , which comprises items (α = . ; e.g., "i am good at resisting temptation"; = not at all like me; = very much like me). self-control was measured in counterbalanced order at the beginning or the end of the questionnaire. descriptive statistics and inter-correlations of all variables are presented in table . inspection of correlations between self-control and self-reported flexibility in the response to covid- crisis showed that people with higher self-control reported a higher overall flexibility, r ( ) = . , p < . . next, we examined whether this perception of flexibility reported by high self-control people is reflected in their ability to continue performing goal-directed behaviors developed before the pandemic and/or develop new behaviors to pursue their goals despite the pandemic-caused disruptions. on average, participants reported . behaviors (sd = . , min = . , max = . ) that they had been using to reach their goals before the lockdown started. the number of behaviors reported was uncorrelated with trait self-control, r = . , p = . . however, during the lockdown, people with higher (vs. lower) trait self-control were more likely to continue engaging in behaviors they had developed to reach their goals prior to the lockdown, r( ) = . , p < . . as our data have two levels, with participants (level ) being able to report multiple behaviors (level ), we additionally analyzed the data using multilevel regression. multilevel regression is particularly suitable here as it accounts for the non-independent data structure (behaviors nested within participants) by modelling participants as random (i.e., including a random intercept at the level of participants; hox, ) . all variables were standardized before the analyses, so that the parameters can be interpreted as standardized coefficients. we used lme package (bates, maechler, bolker, & walker, ) in r. consistent with the correlation analyses reported above, trait self-control was positively associated with the ability to continue performing prepandemic goal-directed behaviors during the lockdown, β = . , p < . . among all participants, . % (n = ) started new behaviors to reach their goals during lockdown. starting new behaviors (dummycoded, = yes) was uncorrelated with trait self-control, r( ) = . , p = . . that is, people with higher self-control were not more or less likely to have developed new behaviors to reach their goals. if we examine only the group of participants who have started new behaviors, we can see that they reported to have started on average . new behaviors (sd = . , min = . , max = . ). moreover, among these participants who developed new behaviors, the number of new behaviors reported was again uncorrelated with trait self-control, r = . , p = . . that is, people with higher self-control were not more or less likely to report more new goal-directed behaviors. however, people with higher self-control found it easier to develop new behaviors, r( ) = . , p = . , and were more likely to report that these behaviors have already become habits, r( ) = . , p = . . multilevel regression results supported these conclusions: self-control was positively associated with the ease of developing new goaldirected behaviors, β = . , p = . , and the perception that the new behaviors have become habits, β = . , p = . . people with higher self-control reported more progress towards their goals, r( ) = . , p < . . we further explored whether the ability to stick to pre-pandemic goal-directed behaviors, the perceived ease of developing new goal-directed behaviors or the ability to turn new behaviors into habits explain high self-control individuals' success at goal attainment. we used parallel mediation analyses with the three variables (average values across the reported goal-directed behaviors) as parallel mediators. the results of the mediation are shown in fig. . the indirect effects of the ability to continue pre-pandemic goal-directed behaviors and to turn newly developed behaviors into habits were significant mediators. that is, trait self-control predicts goal attainment as it is associated with both sticking to pre-pandemic goaldirected behaviors and turning new goal-directed behaviors into habits. developing structures, routines and beneficial habits explains high self-control people's success at goal attainment (adriaanse et al., ; de ridder & gillebaart, ; ent et al., ; galla & duckworth, ; stavrova et al., ; stavrova et al., ) . is this also true during challenging times such as the covid- pandemic? our results showed that self-control passes the 'quarantine test': individuals higher (vs. lower) in self-control made more progress towards reaching their goals during quarantine. this was explained both by continuing prepandemic goal-directed behaviors and by turning newly developed goal-directed behaviors into habits. this finding adds to prior literature by showing that the benefits of high self-control persist under uniquely disruptive circumstances. even though the world had changed dramatically, high self-control people demonstrated a remarkable capacity to stick to pre-existing habits and had the flexibility to develop new habits that better met situational demands. the combination of these two -maintaining past habits and developing new ones -is high self-control people's recipe for success in turbulent times. a practical implication of our findings is that interventions aimed at increasing self-control may be beneficial also in the context of the challenges posed by the covid- pandemic. further research using objective measures of goal attainment, longitudinal data and non-student populations is needed to corroborate these insights. effortless inhibition: habit mediates the relation between self-control and unhealthy snack consumption fitting linear mixed-effects models using lme the psychological impact of quarantine and how to reduce it: rapid review of the evidence lessons learned from trait self-control in wellbeing: making the case for routines and initiation as important components of trait self-control taking stock of self-control: a meta-analysis of how trait self-control relates to a wide range of behaviors trait self-control and the avoidance of temptation more than resisting temptation: beneficial habits mediate the relationship between self-control and positive life outcomes introduction to mediation, moderation, and conditional process analysis: a regression-based approach multilevel analysis: techniques and applications what's so great about self-control? examining the importance of effortful self-control and temptation in predicting real-life depletion and goal attainment covid- and mental health: a review of the existing literature drinking to cope with the pandemic: the unique associations of covid- -related perceived threat and psychological distress to drinking behaviors in american men and women quarantine has changed us -and it's not all bad choosing goals that express the true self: a novel mechanism of the effect of self-control on goal attainment finding meaning in self-control: the effect of self-control on the perception of meaning in life high self-control predicts good adjustment, less pathology, better grades, and interpersonal success life-changing habits you don't want to give up after the pandemic handbook of self-regulation: research, theory, and applications key: cord- - dzqzwx authors: bolzoni, luca; bonacini, elena; soresina, cinzia; groppi, maria title: time-optimal control strategies in sir epidemic models date: - - journal: mathematical biosciences doi: . /j.mbs. . . sha: doc_id: cord_uid: dzqzwx abstract we investigate the time-optimal control problem in sir (susceptible-infected-recovered) epidemic models, focusing on different control policies: vaccination, isolation, culling, and reduction of transmission. applying the pontryagin’s minimum principle (pmp) to the unconstrained control problems (i.e. without costs of control or resource limitations), we prove that, for all the policies investigated, only bang–bang controls with at most one switch are admitted. when a switch occurs, the optimal strategy is to delay the control action some amount of time and then apply the control at the maximum rate for the remainder of the outbreak. this result is in contrast with previous findings on the unconstrained problems of minimizing the total infectious burden over an outbreak, where the optimal strategy is to use the maximal control for the entire epidemic. then, the critical consequence of our results is that, in a wide range of epidemiological circumstances, it may be impossible to minimize the total infectious burden while minimizing the epidemic duration, and vice versa. moreover, numerical simulations highlighted additional unexpected results, showing that the optimal control can be delayed also when the control reproduction number is lower than one and that the switching time from no control to maximum control can even occur after the peak of infection has been reached. our results are especially important for livestock diseases where the minimization of outbreaks duration is a priority due to sanitary restrictions imposed to farms during ongoing epidemics, such as animal movements and export bans. the emergence and re-emergence of infectious diseases represent a major threat to public health and may cause heavy economic and social losses. recent epidemics of ebola in west africa and mers-cov in south korea highlighted once again the requirement for strong public health interventions for fast disease eradication [ , ] . in a similar way, outbreaks of infectious diseases in domestic animals may cause significant consequences for both the sustainability of the livestock industry and the costs associated to disease surveillance, control, and eradication. moreover, the economic burdens imposed by livestock diseases exceed the agricultural compartment, by affecting also commerce, tourism, and even human health in the infected areas. consequently, minimizing the time pe-the time of infection, carrying on until the disease-free status is formally regained [ ] . examples of costly restrictions for the livestock industry include: the export ban of uk cattle because of the bovine spongiform encephalopathy epidemic [ ] and the export ban of poultry and poultry related products in hong kong, laos, thailand, and the netherlands due to outbreaks of highly pathogenic avian influenza [ ] [ ] [ ] . by using a stochastic modeling framework for classical swine fever in the netherlands pig farms, mangen et al. [ ] showed that the increase of the epidemic duration affects the sanitary costs associated to disease outbreaks more than a proportional growth in the number of infected farms. this prediction followed from the observation that longer epidemics are more widespread, involving a larger number of animals slaughtered. the estimate of the epidemic duration appears almost invariably in the simulation outputs of data-driven mathematical models developed to evaluate the effectiveness and the efficiency of surveillance and control policies for several infections in livestock, such as foot-andmouth disease [ ] , classical swine fever [ ] , bovine tuberculosis [ ] , and avian influenza [ ] . however, few attempts have been made to address the problem of minimizing the epidemic duration from a theoretical point of view by using optimal control theory. to our knowledge, the only example of analytic characterization of the control function in a time-optimal framework is due to jiang [ ] , who focused on the analysis of isolation strategies in a subsystem of the model proposed in zhang et al. [ ] to describe sars spread. on the other hand, the optimal control theory has been widely applied to solve the problem of minimizing the total number of infected individuals (or the total infectious burden) in basic sir (susceptible-infected-recovered) epidemic models by means of different control policies, such as: the implementation of emergency prophylactic vaccination plans, the isolation of infected individuals, the reduction of disease transmission through the limitation of contacts between individuals, and non-selective culling [ ] [ ] [ ] [ ] [ ] [ ] [ ] . prophylactic vaccination consists in the vaccination of susceptible individuals; its goal is to prevent the development of diseases. isolation consists in the quarantine of infected individuals. as regards livestock diseases, in sir models isolation is mathematically equivalent to removal of infected individuals through testand-cull procedures. non-selective culling consists in the slaughtering of both infected and healthy individuals and it is usually implemented in wildlife and livestock when no other options are available (e.g. no diagnostic tests available, lack of time or resources). the rationale for culling healthy individuals resides in the positive relationship between the rate at which individuals become infected and the abundance of susceptible individuals. among humans, the reduction of transmission can be obtained through information campaigns or emergency movement bans (e.g. school closures, flight limitations), while in livestock it can be obtained by imposing limitations on animal, vehicle, and personnel movements among farms. the previously cited studies solved the optimal control problem for the minimization of the infectious burden in unconstrained conditions (i.e. without costs of control or resource limitations). they showed that the optimal strategy always relies in the adoption of the maximum control for the entire epidemic. in this context, maximum control is intended as the implementation of the control policy at its maximum available rate. here, by using simple sir models in an optimal control framework [ ] , we thoroughly investigate the problem of minimizing the epidemic duration by using prophylactic vaccination, isolation, non-selective culling, or reduction of transmission controls. in this study, we will show that the optimal control strategies to minimize the epidemic duration in sir models can substantially differ from those minimizing the infectious burden. specifically, we will prove that: ( i ) using the maximum control for the entire epidemic duration may not be the optimal strategy (even in unconstrained conditions); and ( ii ) when the maximum control for the entire epidemic is not an optimal strategy, a delayed control is optimal. consequently, our results lead to the conclusion that minimizing the epidemic duration does not always imply minimizing the total infectious burden, and vice versa. we describe the evolution of the infection in a host population with a standard deterministic sir model [ ] , that can be described by the following system of two ordinary differential equations (odes): where s ( t ) and i ( t ) represent the number of susceptible and infected hosts, respectively, β represents the transmission rate of the infection and μ represents the loss rate of infected individuals through both mortality and recovery. if we denote by x (t) = (s(t) , i(t )) t the column vector that describes the state of the system at time t , we can rewrite system ( ) in the more compact form ˙ in our analysis, we consider four different control policies, namely: vaccination, isolation, culling, and reduction of transmission. we denote the generic control policy rate applied at time t by u ( t ), which is assumed to be a piecewise continuous function that takes values in a positive bounded set u = [ , u max ] . we apply the different policies separately by adding a linear term in the control variable u ( t ) to model ( ) , namely considering the general system ˙ where the function g depends on the chosen control policy. specifically, we define a general linear term policy which is a linear function of s and i that allows to model culling and, in addition, we consider the nonlinear term policy reduction of transmission we define the basic reproduction number for model ( ) as r = βs( ) /μ, which represents the average number of secondary infections produced by a single infected individual in a completely susceptible population in the absence of control [ ] . in addition, for each policy we will define the control reproduction number r c that represents the average number of secondary infections produced by a single infected individual in a completely susceptible population with control measures in place [ ] . from this definition, it follows that, when r c < , control measures applied at the beginning of the epidemic are able to immediately reduce the number of the infected individuals (i.e. ˙ the target will be the minimization of the eradication time of the infection. existence of the eradication time in problem ( ) is guaranteed by the results in appendix a . definition : (eradication time) . the eradication time t of the controlled sir problem ( ) is the first time at which the number of infected individuals reaches the threshold ε, where ε < is a fixed positive constant. we will chose initial conditions of infected individuals i ( ) strictly greater than ɛ . as a consequence, t being the first time at which the variable i reaches ɛ , it holds ˙ we can then write the optimal control problem [ ] where the goal is: subject to: ˙ given the optimal control problem ( ) with f, g ∈ c ∞ ( r ) , we apply the pontryagin's minimum principle [ ] in order to find a characterization of the optimal control strategy. theorem : (pontryagin's minimum principle for linear timeoptimal control problem [ ] ) . suppose that u * ( t ) is a minimizer for the optimal control problem ( ) and let x * (t) = (s * (t ) , i * (t )) t and t * denote the optimal solution of problem ( ) and the optimal eradication time, respectively. then, there exists a piecewise c vector func- where the hamiltonian is defined as h(x , u, λ) = + λ t ( f (x ) + ug(x )) , and: . the function h (w ) = h(x * (t) , w, λ * (t)) attains its minimum on u at w = u * (t) : for every t ∈ [ , t * ] ; . the hamiltonian is constant equal to zero along the optimal solution: . the following transversality condition holds: λ * s (t * ) = . moreover, because the hamiltonian is linear in the control variable, the value of u * ( t ) is determined by the sign of the switching function does not vanish: the results that we will prove in the next sections can be summarized in the following theorem. for each considered control policy the optimal control for problem ( ) is bang-bang. the optimal strategy consists either in a constant control u * (t) ≡ u max or in a delayed control → u max with a single switching time τ * s , namely u * (t) = for t ∈ [ , τ * s ) and u * (t) = u max for t ∈ (τ * s , t * ] . in addition, if the optimal control is delayed, three different behaviors are allowed, depending on the position of the switching time τ * s compared to the peak of infection, leading to the four different types of admissible optimal control sketched in fig. . we will denote the set of such admissible optimal controls by as regards the numerical solution, several numerical methods for the optimal solution of both minimum time and bang-bang control problems can be found in literature. such techniques are mainly based on shooting methods [ ] [ ] [ ] , smooth regularizations of the control function [ ] , or pseudospectral methods [ ] . however, since our problem is characterized by the particular class of optimal controls a in ( ) , for our numerical simulations we will use a simpler ad hoc numerical scheme. the method is based on the idea of identifying each bang-bang function u (t) ∈ a with a real parameter. since the delayed optimal control function is not defined at the switching time instant, for numerical simulations we fix by convention u * (τ * s ) = u max . then, we can generalize the concept of switching time, defined as the zero of the switching function ψ, introducing the starting intervention time which represents the first time instant at which the control u (t) ∈ a assumes the value u max . since a delayed control can be characterized by its switching time τ s , we can then identify each admissible optimal control in a , constant or delayed, by the value of τ and write it in the more general form: therefore, the functional to be minimized j ( u ) can be seen as the function j : τ → t , that links the starting intervention time τ ≥ to the eradication time t of the problem ˙ an optimal control u * will be identified by a starting intervention time such that τ * = argmin j, since it can be proved that j always admits at least a minimum value (see theorem a. ). the numerical solution will be computed by evaluating the function j ( τ ) over a suitable interval and looking for its minimum value. in particular, we fix a uniform mesh { τ i , i = , . . . , m} over the interval [ , t unc ], where t unc is the eradication time of the uncontrolled epidemic. for each mesh point we consider the related control function u ( t ; τ i ) and numerically integrate the cauchy problem ( ) using the crank-nicholson method with uniform time steps { t k , k = , . . . , n} , obtaining the numerical solution then, we set the eradication time t i relevant to the mesh point τ i as the first time step t k at which the computed solution i (i ) k ≤ ε. finally, we take the minimum over the set of computed eradication times t j = min { t i , i = , . . . , m} as the optimal eradication time, and set the corresponding τ j as the optimal starting intervention time. in the following sections, we investigate the four different control policies considered. for each policy we will present theoretical and numerical results. in our numerical simulations, we set ε = . , as in [ ] . then, through a sensitivity analysis, we explore the solutions of optimal control problems ( ) -( ) on a wide range of parameter settings describing different epidemiological conditions (represented by r = βs( ) /μ), different possible control efforts (represented by u max ), and a different number of initially introduced infected individuals in the population (represented by i ( )). we consider sir model ( ) with the general linear term control, denoted by u l ( t ), obtaining an optimal control problem as the one defined in ( ) , with g l ( x ) as in ( ) . l is the optimal control strategy for the linear term control problem, then u * l is a bang-bang control with at most one switching time τ * s from no control to maximum control. proof. see b. . we proceed now to analyze the peculiarities of each policy involved in the general formulation. we consider the vaccination control, denoted by u v ( t ) in the optimal control problem ( ) , with g v ( x ) as in ( ) . the control reproduction number for vaccination is defined as r v for this policy, it is easy to prove that there exists a unique time instant t p (possibly ) at which the function ˙ i changes sign. in particular, ˙ we call t p the peak time, because it represents the time at which the number of infected individuals reaches its maximum. therefore, in addition to the general result of theorem , it is possible to prove the following. theorem . the switch of the optimal control u * v can occur only before the peak of the infection. the numerical analyses on the time-optimal vaccination problem are illustrated in fig. . in fig. (a) , we show the results of the simulations performed in the parameter space as explained in the color codes in fig. , the light gray and v , r (β ) ] for which the time-optimal vaccination problem selects for delayed and constant control, respectively. as highlighted by the analytic results, fig. (a) displays that the switching time always occurs before the peak of infection (light gray region) and that higher vaccination effort s always select f or a constant control. fig. (b) shows the optimal starting intervention time ( τ * ), the eradication time for the optimal vaccination strategy ( t * , solid curve), and the eradication time for the constant vaccination ( t τ = , dashed curve) as functions of the maximum effort, u max v . in fig. (b) , we notice that the optimal starting intervention time undergoes a "catastrophic" transition ( sensu [ ] ) from delayed to constant control for increasing values of u max v . then, small changes in u max v can cause an abrupt change in the starting point of the optimal vaccination campaign. on the other hand, fig. (b) shows that, when delaying the onset of vaccination is optimal, the differences in the final time of the epidemic between optimal control and constant control (i.e. variation in the objective function) is marginal. we consider sir model ( ) with isolation control, denoted by u i ( t ), obtaining an optimal control problem as the one defined in ( ) , with g i ( x ) as in ( ) . the control reproduction number for isolation is defined as r i the numerical analyses on the time-optimal isolation problem are illustrated in fig. . in fig. (a) , we show the results of the simulations performed in the parameter space [ u max i , r (β ) ]. conversely to vaccination, our results show that the time-optimal isolation problem can select for delayed strategies also for high values of maximum effort, u max i . moreover, the switching time for the optimal isolation strategy can occur after the peak of infection (see the dark gray region in fig. (a) ). in fig. (b) , we show that the isolation problem selects for optimal delayed control in a wide range of parameter settings also when the number of infected individuals firstly introduced in the population increases (i.e. i ( ) > ). fig. (c) displays the optimal starting intervention time ( τ * ), the final time for the optimal isolation strategy ( t * , solid curve), and the final time for the constant isolation ( t τ = , dashed curve) as functions of the maximum effort, u max i . as in the vaccination problem, the optimal starting intervention time for isolation undergoes a "catas-trophic" transition from delayed to constant control for increasing values of maximum effort. fig. (c) shows that delayed control can be optimal also when r c < , i.e. when an prompt intervention at t = could have implied an immediate decline in the number of infected individuals. in addition, when delaying the onset of isolation is optimal, the differences in the final time of the epidemic between optimal control and constant control can be significant. we consider the culling control, denoted by u c ( t ), in the optimal control problem defined in ( ) , with g c ( x ) as in ( ) . the control reproduction number for culling is defined as r c the numerical analyses on the time-optimal culling problem are illustrated in fig. . in fig. (a) , we show the results of the simula- . in addition, in the aforementioned cases, the starting of the optimal culling generally occurs before the peak of infection (light gray region in fig. (a) ). however, we can notice that there exists a small region in the parameter space [ u max c , r (β ) ] where the starting of the optimal strategy can occur after the peak of infection (see the dark gray region in the box). fig. (b) shows the optimal starting intervention time ( τ * ), the final time for the optimal culling strategy ( t * , solid curve), and the final time for the constant culling ( t τ = , dashed curve) as functions of the maximum effort, u max c . also in this case the optimal starting intervention time undergoes a "catastrophic" transition from delayed to constant control for increasing values of u max c and, when delaying the onset of culling is optimal, the differences in the final time between optimal control and constant control is marginal, analogously to the case of vaccination. we consider sir model ( ) with reduction of transmission control, denoted by u r ( t ), obtaining an optimal control problem as the one defined in ( ) , with g r ( x ) as in ( ) and < u max r ≤ . the control reproduction number for reduction of transmission is defined as r r c = β( − u max r ) /μ. despite the nonlinear term included in the reduction of transmission policy, it is possible to find the same type of optimal strategy of the linear term policies. theorem . if u * r is the optimal control strategy for the reduction of transmission problem, then u * r is a bang-bang control with at most one switching time τ * s from no control to maximum control. the numerical analyses on the time-optimal reduction of transmission problem are illustrated in fig. . in fig. (a) , we show the results of the simulations performed in the parameter space [ u max r , r (β ) ]. we display that, when delayed control is selected, the starting of the optimal reduction of transmission generally occurs after the peak of infection (dark gray region in fig. (a) ). in fig. (b), we show that the reduction of transmission problem selects for optimal delayed control in a wide range of parameter settings also when the number of infected individuals firstly introduced in the population increases (i.e. i ( ) > ). similarly to the isolation problem, we find that: ( i ) delayed control for reduction of transmission can be optimal also when r c < ; and ( ii ) when delaying the starting of reduction of transmission is optimal, the differences in the final time of the epidemic between optimal control and constant control can be significant. similarly to the isolation problem, s ( t * ) exhibits a discontinuous increase at the boundary between delayed and constant control. in this work, we investigated the problem of minimizing the epidemic duration by using different control policies. specifically, we characterized analytically the time-optimal control strategies for prophylactic vaccination, isolation, non-selective culling, and reduction of transmission by using a family of simple sir models in an optimal control framework [ ] . our analyses led to the nontrivial result that, even in the unconstrained optimal control problem (i.e. without costs of control or resource limitations), using the maximal effort for the entire epidemic period may not be the optimal strategy to minimize the epidemic duration. in addition, we found that, when applying the maximal effort for the entire epidemic is sub-optimal, then a delayed control represents the optimal strategy in all the cases investigated. we even found that the optimal amount of delay applied to the control may be sufficiently large to postpone the beginning of the intervention after the peak of the infection (see fig. and dark gray regions in figs. (a), (a) , and (a)). in addition, we showed that the delayed control may represent the optimal strategy for minimizing the epidemic duration even when a prompt intervention could immediately reduce the number of infected individuals (i.e. reduce r c below , see figs. (c) and (c)). the biological explanation for the optimality of delayed controls relies on the remark that, at the beginning of the epidemic, the in- fection process can be more efficient in depleting the reservoir of susceptibles (which represents the mechanism leading to epidemic extinctions) than the applied control. in other words, reducing via external interventions the number of individuals involved in the infection process at the beginning of the outbreak (especially the infected ones) may lead to slower epidemic dynamics, which implies longer times for the epidemic to go extinct. two evidences support this explanation: ( ) delayed control is generally optimal when the effectiveness of the control is low (i.e. low u max ); and ( ) isolation and reduction of transmission policies (which do not reduce directly the number of susceptibles) tend to select for delayed control in wider ranges of parameter settings than vaccination and culling. our results differ from those previously obtained for the timeoptimal problem in specific epidemic contexts. by analyzing a subsystem of an epidemic model describing sars spread, jiang [ ] proved that, according to pontryagin's minimum principle, maximizing the isolation effort f or the entire epidemic period would reduce epidemics in minimum time. similarly, by numerically testing scenarios in an sir model where the control always reduces the disease reproduction number below , iacoviello & liuzzi [ ] showed that maximizing the combined vaccination and isolation effort s f or the entire epidemic period eradicates epidemics in minimum time. our results substantially differ also from those obtained minimizing the total number of infected (or the infectious burden) in sir epidemic models. by characterizing optimal controls according to pontryagin's minimum principle, different works showed that the unconstrained problems for isolation [ , ] , vaccination [ , ] , and culling [ ] only support the trivial solution of applying the maximal effort for the entire epidemic. then, from our results it follows that the infectious burden may not be minimized while minimizing the epidemic duration in simple sir models. minimizing the infectious burden in the optimal control problem for isolation and reduction of transmission is equivalent to maximize the final number of susceptibles, s ( t ). some examples of the tension between minimizing the epidemic duration and the infectious burden can be observed in figs. and . in particular, figs. (c) and (c) display the eradication time, t , and figs. (d) and (d) display the number of susceptible individuals at the end of the epidemic, s ( t ), as functions of u max for both the timeoptimal control and the constant control (corresponding to the optimal solution for the unconstrained problem of infectious burden minimization). from these figures, we notice that the differ-ent objective functions provide similar results when the control effort s are sufficiently large to rapidly lead the epidemic to extinction (high u max ), while they provide substantially different results in the case of less efficient strategies (low u max ). specifically, the time-optimal control strategy performs poorly in minimizing the infectious burden at the boundary between delayed and constant control (see figs. (d) and (d)), while the infectious burden minimization strategy performs poorly in minimizing the epidemic duration for slightly higher values of r c (see the peak of t τ = in figs. (c) and (c) ). moreover, we find that small changes in the control parameter u max can cause large changes in the shape of the optimal strategies. an analogous result was found by hansen & day [ ] investigating the problem of minimizing the infectious burden through isolation in a sir framework with limited resources. hansen & day [ ] also found that a "catastrophic" shift in the shape of the isolation strategy corresponds to an abrupt variation in the objective function (i.e. the infectious burden). conversely, here we find that "catastrophic" shifts in the shape of the control strategies correspond to continuous variations in the objective functions (i.e. the final time of epidemics). we believe our findings can be useful in throwing light on overlooked results obtained with more complex models developed in specific epidemiological contexts. for instance, roche et al. [ ] investigated the performances of different spatially explicit models for the spread of foot-and-mouth disease in the uk farms, considering different control scenarios. among other scenarios, they compared the effect of suppressive vaccination strategies started at and days after the outbreak beginning. they found that, in two out of the four models investigated, the medians and/or the th percentiles of the epidemic duration decreased when the control is delayed by days [ , see models 'is+' and 'nl' in table therein]. on the other hand, they found that the number of infected farms always increases when the vaccination is delayed [see table in ] . in a similar way, by investigating the effectiveness of combined culling and movement restriction to control classical swine fever in switzerland pig farms, dürr et al. [ ] found that delaying the starting of the control from to days after the outbreak beginning reduced the median outbreak duration in three out of the eight analyzed scenarios [see fig. in ]. previous works have already shown that delayed control might represent an optimal strategy in some epidemiological applications. for instance, handel et al. [ ] and hansen & day [ ] showed that delaying the controls may be optimal in preventing the re-emergence of the epidemic or the emergence of resistant epidemics. bolzoni et al. [ ] showed that the delayed control may be optimal in wildlife diseases where the host population growth is density-dependent. the numerical analyses performed here under the assumption of constant control highlighted that increasing the control effort s may lead to a substantial increase of the eradication time. this is especially true in the case of isolation and reduction of transmission, where the eradication time may increase from two-to fivefold with respect to the "do-nothing" alternative (see figs. (c) and (c)). similar negative effects of constant efforts on disease control have also been highlighted when the target of the intervention was the reduction of the number of infected individuals [ ] [ ] [ ] [ ] . all these counter-intuitive findings suggest that the implementation of simple time-dependent strategies may crucially improve the control of infectious diseases. other aspects of diseases control implementation that were not included in the present work -such as combined controls [ ] , the costs of control [ ] , resources limitation [ ] , and availability of surveillance information [ ] -can play a significant role in shaping the optimal strategy. these aspects are essential in defining optimal protocols of intervention for diseases eradication. how-ever, thanks to the generality of the model formulation, we believe our results can be used as a landmark for future investigations in the directions listed above. proof. by definition, there exists an optimal solution of ( ) if the functional j ( u ), which gives the eradication time of the controlled sir problem ( ) as a function of the control, has (at least) a minimum point u * on the set of admissible controls. for each policy theorem holds, namely the set of admissible controls is a given in ( ) . as detailed in section , we can see j as a function that links the starting intervention time τ ( ) to the eradication time t : since the starting intervention time cannot be larger than the eradication time of the uncontrolled epidemic, then there exists an upper bound τ max for τ . we prove that j admits at least a minimum point τ * by proving that it is a continuous function on the bounded interval [ , τ max ] . first we prove that j is a continuous function in , namely that where y is the solution of the uncontrolled problem ˙ y by the continuous dependence on initial conditions theorem, for a generic t ≥ h it holds: being a continuous positive function that is strictly monotone in a neighborhood of t , it is invertible and therefore we can state . the proof of the continuity of j in a generic starting intervention time τ follows from the continuity in , using translation arguments. remark a. . if we consider non-negative initial conditions s ( ) and i ( ), then the solution of the differential system ( ) is nonnegative at each time t > . indeed, for all the chosen policies, the i axis is a trajectory for the system; the s axis is also a trajectory (for vaccination and culling policies) or is a set of stationary points (for isolation and reduction of transmission policies). using results of remark a. it is sufficient to prove that for s, i > the vector field evaluated on the boundary line s + i = k points towards the internal part of the region q k [ ] ; it is straightforward for each policy since the scalar product of the vector field f ( x ) and the outward pointing normal vector of the boundary ˆ n = ( , ) t is negative in all cases. throughout all the proofs we omit the superscript * for the optimal quantities, in order to simplify the notation. let x (t) = (s(t ) , i(t )) t denote the optimal solution for the control problem with linear term policy, suitable to model vaccination, isolation or culling for proper values of parameters α and α ; let u l ( t ) be the control term, λ(t) = (λ s (t ) , λ i (t )) t the corresponding adjoint variables and t the optimal eradication time. by the pontryagin's minimum principle, the hamiltonian function, the switching function and its derivative are respectively: and the adjoint variables satisfy the following system of odes: ) the sketch of the proof of theorem is as follows. (i) first we prove that the control is non-singular, namely that the function ψ vanishes only in isolated points. suppose in fact that ψ vanishes in an open interval b . then also all the derivatives vanish there and in particular ψ = ˙ ψ = in b , which yields by some algebra λ s = λ i = by (b. ) , since s, i > when s ( ), i ( ) > (see remark a. ). this is in contradiction with theorem , as the adjoint variables λ s and λ i cannot vanish simultaneously by construction, therefore ψ vanishes only in isolated points. as a consequence, the control is a piecewise constant function u l ( t ) that can assume only two values: and u max . the switching times are defined as the time instants at which the function ψ( t ) changes its sign and, consequently, the function u l ( t ) changes its value. therefore two types of switch can occur: either the value of u l ( t ) is in a left-neighborhood of the switching time and is u max in a right-neighborhood, and we denote it by → u max , or the converse, which is denoted by u max → . (ii) next we show that the optimal control in a left-neighborhood of the eradication time t must be equal to u max . by con- of theorem , we can write λ s , and therefore λ i and ˙ ψ , as functions of in particular, we can see that the sign of ˙ ψ is opposite to the sign of q . suppose that there are multiple switching times τ ( j) s , j = , . . . , n . we have already proved that u l (t ) = u max , so at the last switching time ˙ ψ (τ (n ) s ) < must hold, thus q (τ (n ) s ) > . since q is a decreasing function, this means that q is positive in the interval of the function ψ after the peak is then from negative to positive, which is not admissible, since we proved that ψ is negative in a left-neighborhood of t . thus the switch can only occur before the peak and, being ˙ ψ (τ s ) always negative, it must be unique. moreover, since ψ changes its sign from positive to negative values, the control switches from to the maximum rate u max . the sketch of the proof of theorem is as follows. (ii) by definition of the basic reproduction number, we know that if r < then the number of infected individual is monotonically decreasing in time, namely the peak of the infection is t p = . as we already proved that there cannot be a switch for t > t p , in this case the optimal control must be u v (t) ≡ u max v . suppose that r > and that the optimal control is delayed with switch → u max v at time τ s > . then we prove that the relation μ > u max v must hold. first we prove that, under those hypotheses, the function ψ has a minimum point m ψ in ( τ s , t ) at which λ i ( m ψ ) < λ s ( m ψ ), as sketched in fig. b. . the function ψ vanishes at τ s (by definition) and at t (by the transversality condition), while it is strictly negative between the two points, therefore it must have at least a minimum point. since ψ is a c function, in such points ˙ ψ = and thus also λ i = , by (b. ) . substituting this latter value in the second derivative of the switching function and recalling 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optimal culling strategy to control infectious diseases in wildlife the mathematical theory of optimal processes, international series of monographs in pure and applied mathematics population biology of infectious diseases: part i computational method for time-optimal switching control chapman & hall using switching detection and variational equations for the shooting method smooth regularization of bang-bang optimal control problems a modified pseudospectral scheme for accurate solution of bang-bang optimal control problems stabilité structurelle et morphogénèse, essai d'une théorie générale des modèles fixed/free final time sir epidemic models with multiple controls what is the best control strategy for multiple infectious disease outbreaks optimal antiviral treatment strategies and the effects of resistance harvesting can increase severity of wildlife disease epidemics transmission heterogeneity and control strategies for infectious disease emergence wildlife disease elimination and density dependence unexpected consequences of culling on the eradication of wildlife diseases: the role of virulence evolution a cost analysis of alternative culling strategies for the eradication of classical swine fever in wildlife nonlinear oscillations, dynamical systems, and bifurcations of vector fields the authors are very grateful to stefano pongolini for his invaluable suggestions and remarks that improved the manuscript. lb was supported by the italian ministry of health (grant izsler prc ). support by indam-gnfm is also gratefully acknowledged by eb, cs and mg. appendix a theorem a. . there exists an optimal solution of the optimal control problem ( ) . key: cord- - tf ah g authors: weber, wilfried; fussenegger, martin title: emerging biomedical applications of synthetic biology date: - - journal: nat rev genet doi: . /nrg sha: doc_id: cord_uid: tf ah g synthetic biology aims to create functional devices, systems and organisms with novel and useful functions on the basis of catalogued and standardized biological building blocks. although they were initially constructed to elucidate the dynamics of simple processes, designed devices now contribute to the understanding of disease mechanisms, provide novel diagnostic tools, enable economic production of therapeutics and allow the design of novel strategies for the treatment of cancer, immune diseases and metabolic disorders, such as diabetes and gout, as well as a range of infectious diseases. in this review, we cover the impact and potential of synthetic biology for biomedical applications. | mammalian gene expression control strategies. a | repression-based expression control. a repressor protein binds to its operator and thus prevents activation of the promoter and expression of the gene of interest. in response to an inducer, the repressor dissociates from the operator, the promoter is derepressed, and the gene of interest is expressed. b | activation-based expression control. a minimal promoter (p min ) is activated when a chimeric transcription factor that is constructed by fusing a repressor protein to a transcription activation domain binds to its operator. in the presence of an inducer, the repressor protein-transcription-activation domain complex dissociates from its operator, p min is no longer activated, and transcription of the gene of interest is prevented. c | mrna transcript-based expression control. a self-cleaving ribozyme is fused to a small-molecule-binding aptamer and introduced into the ′ untranslated region (utr) of a gene of interest. in the absence of the inducer, the ribozyme undergoes self-cleavage, thereby eliminating the poly(a) tail (pa) from the open reading frame and preventing translation. however, in the presence of the inducer, the aptamer undergoes a conformational change, which inactivates the ribozyme and allows translation to occur. genetic circuits that can be switched between two stable expression states (for example, an 'on' and an 'off' state) by a transient stimulus. in the absence of a switching stimulus, the expression state is locked and inherited across cell generations. genetic circuits whose response dynamics depend on a combination of past and present states. the capacity of a species to sense and score its surrounding ecosystem, for example, to identify the type and population density of neighbouring species. a small-molecule-based chemical language by which bacteria communicate within and across populations (the 'quorum'). production and response to quorum-sensing molecules is correlated with population density. these are devices that are selectively induced within a specific concentration range of the input signal. at lower or higher trigger levels, the band-pass filter shuts down output signals. are starting to emerge [ ] [ ] [ ] . this review focuses on the recent progress in synthetic biology towards improving human health, including diagnostic applications, design of novel preventive care strategies, progress in drug discovery, design and delivery, and development of novel treatment strategies, such as prosthetic networks. synthetic biology holds the promise of providing unique opportunities for major advances in the improvement of human health in the twenty-first century , , . understanding disease mechanisms pathogen mechanisms. the availability of affordable technology for synthesizing and assembling sequences for proteins or for viral and bacterial genomes with increased speed has dramatically improved our understanding of host-pathogen interactions and of disease mechanisms. the synthetic biology principle of 'analysis by synthesis' provides mechanistic insight by combining rapid synthesis, assembly, shuffling and mutation of individual genetic components with straightforward liposomes that are decorated with antigenic peptides or proteins that elicit an immune response. these are molecular devices that promote the spreading of a specific gene throughout a target population by taking advantage of a mechanism that multiplies the specific gene in the host genome. gene drive systems produce non-mendelian patterns of inheritance. functional analysis. for example, the genome of the h n virus that was responsible for the spanish influenza pandemic was synthesized using sequence information from genomic pieces that were extracted from permafrost-conserved tissue samples. functional analysis of the reconstructed virus provided new insight into the key virulence factors of the pathogen: namely, a haemagglutinin variant that induces membrane fusion without trypsin activation and a modified polymerase that enhances viral replication . the same study also revealed that a combination of eight genes was responsible for the exceptional virulence of the spanish influenza strain , . this finding may help to identify the pandemic potential of future virus variants [ ] [ ] [ ] . synthesis and analysis of chimeric viruses have also made a substantial contribution to the understanding of coronavirus zoonoses that were responsible for the severe acute respiratory syndrome (sars) pandemic of and . the characterization of the history of the sars coronavirus, especially its switch in tropism, was particularly challenging, as its direct ancestors could not be propagated in laboratory models. however, after a kb sars-like bat coronavirus was designed to contain the receptor-binding spike protein of its human homologue, the synthesized chimeric virus was able to replicate in culture and infect mice . these in vivo studies revealed infectionenhancing mutations in the spike protein and established this surface protein as a key factor that is responsible for tropism switches in coronavirus zoonoses . reconstruction of pathogens by dna synthesis can also be used for the production of diagnostic high-density antigen arrays , , such as those used to profile post-lyme-disease syndrome or the humoural immune responses to hepatitis c and the human immunodeficiency virus (hiv) . immune systems. synthetic biology has recently provided new insight into disorders that are related to deficiencies of the immune system, which is known for its particularly complex control circuits and cellular interaction networks. for example, dysfunction of b-lymphocyte activation underlies several physiological disorders . functional reconstitution and analysis of the human b cell antigen receptor (bcr) signalling cascade in insect cells revealed that bcrs are not activated by antigen-specific crosslinking, as presented in textbooks, but instead have an autoinhibitory oligomeric conformation on resting b lymphocytes that shifts to an active dissociated form when antigens bind . this triggers the signalling cascade, which results in antibody production and the onset of a humoural immune response. also, construction of a representation of the complete human peptidome engineered for display on the surface of t phages enabled church and colleagues to discover new autoantigens. they used patient-derived autoantibodies to enrich autoantigenic peptides displayed on the phages; they could then identify the antigens by high-throughput sequence analysis . knowledge of the antigens that are involved in autoimmune processes is important for understanding disease aetiology, developing accurate diagnostic tests and designing drugs that neutralize autoreactive immune cells . vaccines. high-throughput and high-precision assembly and engineering of entire genomes from welldefined genetic components using synthetic biology principles has provided new opportunities for the design of attenuated pathogens for use as vaccines. for example, primates immunized with virus-like particles that were produced by selective expression of particular chikungunya virus (chikv) structural proteins were protected against viraemia after a high-dose challenge; even immunodeficient mice that were treated with monkey-derived antibodies survived subsequent lethal doses of chikv . dna synthesis and assembly has also played an essential part in pioneering a safe live vaccine against the poliovirus . the poliovirus was attenuated by systematic genome-scale changes of adjacent pairs of codons from over-to underrepresented codon sets in viral capsid genes (for example, gcc|gaa is strongly under-represented compared with gca|gag, although both encode ala-glu). these changes reduced translation and impaired the replication competence and infectivity of the virus. this attenuated poliovirus provides protective immunization in mice and offers a high safety standard given the low probability that all individual changes will revert and thus reconstitute infectious wild-type viruses. the genome-engineering approach used here could represent a general strategy for designing live vaccines against infectious diseases. other promising vaccination concepts include using antigen-producing immunostimulatory liposomes as genetically programmable synthetic vaccines and the production of heat-stable oral algae-based vaccines to protect against staphylococcus aureus infections , . vector control. suppression of insect vector populations using transgenic viral strains that harbour conditional dominant-lethal synthetic circuitry may control the transmission of malaria parasites and dengue viruses and could eventually control the spread of untreatable diseases [ ] [ ] [ ] . mosquitoes that are transgenic for a tetracycline-dependent transactivator (tta) that is exclusively expressed in the female's indirect flight muscle can only be propagated in the presence of tetracycline, which represses the transcription of this gene. however, the absence of tetracycline leads to the development of a female-specific flightless phenotype , . putting the eggs of this transgenic mosquito into the ecosystem results in male-only releases; female mosquitoes remain grounded and cannot feed, mate or take blood meals, which effectively represents a lethal phenotype. males do not transmit the disease, but they disseminate the synthetic circuit across the resident wild-type mosquito population , (fig. a) . similarly, a synthetic homing endonuclease-based gene drive system could be used to spread genetic modification, such as malaria resistance, from engineered mosquitoes to the field population. homing endonucleases typically produce a single sequence-specific double-strand break in the host genome that is repaired by homologous recombination using the homing endonuclease gene (heg) as a template. consequently, × × × figure | synthetic biology for understanding and preventing disease. a | a female-specific dominant-lethal gene network for mosquito control. mosquitoes were engineered to express an intron-containing variant of the tetracycline (tet) transactivator (tta) under the control of a flight-muscle-specific promoter (p fm ). in male mosquitoes, the intron is not spliced out, which prevents correct tta translation. in female progeny, however, functional tta translation is restored by sex-specific mrna splicing. this results in the activation of the tta-responsive promoter p tet and the expression of a toxic gene that triggers a flightless phenotype. if mosquitoes are raised in the presence of tetracycline (tet), tta is prevented from activating p tet , which results in a normal phenotype. however, following their release into the tet-free environment, engineered males mate with wild-type females. this transmits the female-specific dominant flightless phenotype and should eventually result in the reduction or extinction of the wild-type population. b | propagation of a selfish gene converting a heterozygous into a homozygous host. the homing endonuclease i-scei is expressed and cleaves its cognate restriction site (rs) on the homologous chromosome. following end resection and repair, the i-scei expression cassette is inserted into the second chromosome. pa, poly(a) tail. these constitute a group of secondary metabolites produced through linear decarboxylative condensation of acetyl-coa with several malonyl-coa-derived extender units to a polyketide chain. many pharmacologically active compounds, such as antibiotics and anti-cancer drugs, belong to the polyketide class. the selfish heg is copied to the broken chromosome in a gene conversion process referred to as 'homing' . expressing the heg i-scei under the control of a male germline promoter enabled efficient homing in transheterozygous males and rapid genetic drive, which led to heg invasion in caged mosquito populations (fig. b) . by engineering the sequence specificity of other hegs (for example, i-anii or i-crei), the gene drive concept could, in principle, be used to knock in or knock out gene functions that target the mosquito's ability to serve as a disease vector . field tests of release of insects carrying dominant lethals (ridl) technology using first-generation ttatransgenic mosquitoes have already been conducted in grand cayman. first, a small-scale release confirmed that transgenic males could survive, mate with wild females and produce transgenic larvae, and then the full field trial showed an % reduction in the numbers of wild mosquitoes about weeks after release. as the study site was not isolated and the surrounding areas contained high densities of wild-type mosquitoes, scoring the actual suppression efficiency remains challenging . drug development drug discovery. synthetic mammalian transcription circuits consisting of a chimeric small-molecule-responsive transcription factor and a cognate synthetic promoter were originally designed for future gene-based therapies, and the aim was to adjust therapeutic transgene expression in mammalian cells in response to a pharmacologically active substance , , , . as most chimeric transcription factors are derived from repressors that manage drug resistance in bacteria (for example, resistance to antibiotics ) and are promiscuous for structurally related compounds, mammalian cells containing such circuitry could also be used in 'reverse mode' , as integrated screening devices for the class-specific discovery of new drug candidates , (for example, new antibiotics ) (fig. a) . when mammalian cells that are transgenic for the screening circuit are exposed to a compound library, they detect and modulate reporter gene expression in the presence of a non-toxic, cellpermeable and bioavailable molecule that has a classspecific core structure and corresponding drug activity (for example, antibiotic activity) (fig. b) . using the same screening setup, compounds have been detected that lock the transcription factor onto the dna, which may block induction of antibiotic resistance in pathogens and render them drug-sensitive (for example, see bioversys). using such compounds alongside the specific antibiotic may offer novel anti-infective treatment opportunities and a new life cycle for established antibiotics (fig. c) . other trigger-inducible transcription control systems can be used in this manner as well, such as those that are responsive to streptogramin , tetracycline or macrolide antibiotics , anti-diabetes drugs or immunosuppressive lactones , . one example of the efficacy of a transcription circuit system involves the bacterial transcriptional repressor ethr. ethr represses transcription of etha and so prevents etha-mediated conversion of the lastline-defence antibiotic ethionamide into a pathogenkilling metabolite . the chemical -phenyethylbutyrate, best known for its strawberry flavour, was the first compound found that specifically inactivated ethr and so triggered etha expression and re-established the sensitivity of mycobacterium tuberculosis to ethionamide (fig. d) . further work revealed other ethr-inactivating ethionamide booster compounds; these have also been successfully tested in a mouse model of human tuberculosis . restoring drug sensitivity by pharmacological inhibition of master resistance regulators may be widely applicable . a further example of the use of synthetic circuitry for drug discovery is provided by mammalian cells that are conditionally arrested in the g phase of the cell cycle by circuitry controlling the expression of the cycline-dependent kinase inhibitor p . these cells reproducibly formed a mixture of isogenic subpopulations of proliferation-inhibited cells and proliferating cells that had spontaneously escaped the synthetic cell cycle block . these cells could be used as a cell-based cancer model and could be used to screen for anticancer compounds that selectively eliminate proliferating cells while leaving arrested ones intact , . drug production and drug delivery. the synthetic pathways that are created by assembling enzymatic cascades or networks in bacteria, yeast and plants have been instrumental for the large-scale economic production of high-value drug and drug precursor compounds, as well as for the biosynthesis of new secondary metabolites with novel therapeutic activities. examples include complex polyketides , , halogenated alkaloids [ ] [ ] [ ] and the precursors of the anti-malaria drug artemisinin (which is produced by the company amyris, for example) and of the anti-cancer compound taxol . for production of these compounds, it was necessary to overcome several challenges, including the functional expression of complex biosynthetic enzymes (such as cytochrome p monooxygenases ) and the overall orchestration of the multistep pathway to avoid accumulation of (toxic) intermediate products and to ensure metabolic channelling . small-molecule-responsive protein-protein and protein-dna interactions that are used to pioneer gene switches in mammalian cells , , have also been successfully re-engineered in the design of triggerinducible biohybrid materials for drug delivery [ ] [ ] [ ] [ ] [ ] . using synthetic protein-polyacrylamide and dnapolyacrylamide monomers, hydrogels can be produced that dissolve when specific ligands are supplied (fig. ) . biopharmaceuticals (for example, vascular endothelial growth factor (vegf)) supplied during gel formation are loaded into the hydrogel and can be released in a dose-dependent manner after subcutaneous implantation into mice and oral administration of the trigger compound . it is thought that any trigger-inducible protein-protein and protein-dna interactions could be used to produce drug-sensing and drug-releasing hydrogels , , . in chinese hamster ovary (cho-k ) cells, the streptogramin-responsive repressor (pip) was expressed by a constitutive promoter (p const ). pip binds to its multimeric operator (pir ) and represses expression of the reporter gene secreted alkaline phosphatase (seap). exposing this screening cell line to a small molecule library only resulted in seap production for compounds that were streptogramin-like, cell-permeable and non-toxic (indicated by the brown star) . b | discovery of small molecules that are able to overcome antibiotic resistance. the mycobacterium tuberculosis antibiotic resistance regulator (ethr) was fused to the herpes-simplex-derived transcriptional activator (vp ) and expressed in human embryonic kidney cells (hek -t) under the control of a constitutive promoter (p const ). when ethr-vp binds to its cognate operator (o ethr ), the minimal promoter (p min ) is activated, which results in expression of the reporter gene seap. a screen is performed to identify a cell-permeable, non-toxic molecule (indicated by the yellow star) that prevents ethr binding to o ethr , stopping seap expression. c | overcoming resistance to ethionamide in m. tuberculosis. in m. tuberculosis, ethr represses transcription of both the baeyer-villiger monooxygenase (etha) and itself in a negative feedback loop. when -phenylethylbutyrate (indicated by the pink star) is added, it prevents ethr binding its target promoter (labelled 'p' in the figure). this derepresses etha production, thereby turning ethionamide into a cytotoxic compound that kills the mycobacterium. pa, poly(a) tail. interactive biohybrid material based on the interaction of a repressor protein with its cognate dna operator motif. homodimeric tetracycline repressor (tetr) is converted into a single-chain repressor (sctetr) by connecting two tetr subunits through a flexible peptide linker, and it is tagged with six histidines (sctetr-his ). this molecule is coupled to a polymer and is mixed with a polyacrylamide that has copies of a tetracycline operator (teto) attached to it. sctetr binds to teto so crosslinks are formed, making a hydrogel. when tetracycline is added, sctetr releases teto, and the gel is dissolved. this can be used to release another molecule that was attached to the polymer -in this case, the cytokine interleukin (il- ). genetically encoded repair program protecting against dna damage. in prokaryotes, the repair program is coordinated by lexa and reca. dormant individual cells within a bacterial population that show a high tolerance to antimicrobials. biofilms are surface-associated bacterial communities that are encased in a hydrated extracellular polymeric substance (eps) matrix that is composed of polysaccharides, proteins, nucleic acids and lipids. they are crucial to the pathogenesis of many clinically important bacteria and exhibit resistance both to the immune system and to antimicrobial treatments, making them difficult to eradicate , . collins and colleagues successfully engineered bacteriophage t to constitutively express dspb: an enzyme that hydrolyses β- , -n-acetyl-d-glucosamine, which is an adhesin that is required for biofilm formation and integrity in staphylococcus spp. and escherichia coli clinical isolates. the initial infection of a bacterial biofilm with this bacteriophage (known as t dspb ) results in rapid multiplication of the phage and expression of dspb. following lysis, t dspb and dspb are released into the biofilm, which leads to re-infection and degradation of β- , -n-acetyl-d-glucosamine. during the process of t dspb infection, bacterial biofilm cell counts are reduced by . % -over two orders of magnitude greater than when a non-enzymatic phage is used . in a follow-up study, bacteriophage m was engineered to express lexa , which suppresses the sos dna repair system that bacteria require to counteract antibiotic-induced oxidative stress [ ] [ ] [ ] . infection by this designer phage sensitizes e. coli to quinolone antibiotics. use of this phage increases the survival of mice that are infected with e. coli, decreases the survival of antibiotic-resistant bacteria, persister cells and biofilm cells and reduces the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population. it also acts as a strong adjuvant for other bactericidal antibiotics . the designer phage platform can be used to produce other antibiotic adjuvants . although it was once abandoned after the introduction of antibiotics, phage therapy is currently being revisited in several clinical trials around the world as the prevalence of multidrug-resistant pathogens is dramatically increasing. although phage therapy may face clinical challenges associated with development of bacterial phage resistance, phage neutralization by the immune system and pharmacokinetics, the field will certainly receive an impetus from designer phages . bacteria can communicate with each other using a chemical language known as quorum sensing. individual bacteria produce and secrete signalling molecules (called autoinducers) that are common to multiple species or are species-specific. these molecules accumulate as the population grows and can bind to receptors that coordinate colony-wide gene expression or manipulate the behaviour of other bacterial populations. for example, vibrio cholerae produces cholera autoinducer (cai- ) and autoinducer (ai- ), which trigger repression of key virulence factors. feeding infant mice with a probiotic e. coli that naturally produces ai- and has been engineered to constitutively synthesize cai- significantly increased the animals' survival rate after ingesting v. cholerae . this suggests that such an approach could be an economic strategy to prevent infectious diseases. unlike antibiotics, quorumsensing-based interventions do not kill pathogens but reprogram their behaviour; this strategy may be free of selection pressure and therefore may be less prone to develop resistance. in another study, commensal bacteria were equipped with synthetic circuitry to stimulate glucose-dependent insulin production in intestinal epithelial cells . after intravenous injection, escherichia coli accumulates in cancer tissue, where it reaches high population densities. e. coli is engineered to link the quorum-sensing receptor luxr to an autoinducer (ai- )-inducible promoter (p lux ). p lux is also used to drive luxi and the invasin gene inv. luxi produces ai- , generating a positive feedback loop that coordinates invasion throughout the population. b | acetylsalicylic acid (aspirin)-triggered killing of cancer cells after invasion of salmonella spp. salmonella spp. naturally invade cancer cells after intravenous injection. salmonella spp. were engineered with a pseudomonas putida-derived signal-amplifying two-level cascade in which nahr controls salicylate promoter (p sal )-driven xyls expression and xyls then triggers a xyls -dependent promoter (p m )-driven expression of the cytosine deaminase (labelled cd in the figure) . salicylate induces both nahr-based p sal and xyls -mediated p m activation. mammalian cells are resistant to -fluorocytosine because they lack cytosine deaminase, which converts -fluorocytosine into the toxic cancer therapeutic -fluorouracil. c | invasive bacteria suppress oncogene expression. e. coli is engineered to constitutively co-express a catenin β- -specific short hairpin rna (shrna), listeria monocytogenes listeriolysin (llo*) and inv under control of the bacteriophage t promoter (p t ). they invade cancer cells (using the inv protein), escape from the phagosome (using llo*) and knock down the catenin β- oncogene (using shrna). d | therapeutic protein transduction. lentiviral particles are produced using an integrase-negative helper vector (designated 'helper' in the figure) and a constitutive expression vector encoding the protein of interest (designated 'protein' in the figure) fused to viral protein r (vpr) and a protease cleavage site (pc). this can be delivered to any target cell in the absence of viral nucleic acids and proteins. an example application is described in the main text. pa, poly(a) tail. p ef a , elongation factor alpha (ef a) promoter. the action of drugs in the body over a period of time. it covers absorption of the drug as well as its distribution, tissue localization, biotransformation and excretion. commensal bacteria live in close contact with the host. in this special type of symbiosis, one partner is benefited, whereas the other is neither benefited nor harmed. despite decades of progress in cancer therapy, a major challenge remains: how to specifically target and selectively kill neoplastic cells that develop within native and implanted tissue and relocate within the organism to form metastasis. therefore, therapeutic strategies that are designed to eliminate cancer cells must be extremely precise to exclusively target diseased tissue while leaving normal tissue intact. although native cytotoxicity or the constitutive expression of anticancer compounds have demonstrated some potential in animal studies and human clinical trials , trigger-inducible drug expression circuits delivered by tumour-invasive bacteria or tumour-transducing viral particles may improve cancer therapy. synthetic biologists have recently designed a few anti-cancer devices that provide precise timing, location and dosing of drug production by external cues and could provide greater intra-tumoural effects while minimizing systemic toxicity. after intravenous injection or oral administration, many bacterial species (for example, e. coli and salmonella spp.) naturally sense and selfpropel towards tumours. these bacteria have also been engineered to selectively invade and proliferate in tumour tissues and to produce cytotoxic compounds as well as reporter proteins for non-invasive follow-up monitoring of tumour regression . these bacteria express flagella to penetrate tissue and chemotactic receptors to promote migration towards aspartate produced by viable cancer cells, ribose released by necrotic tissue or hypoxic regions generated by the hyper-metabolic activities of neoplastic cells. after they have reached the tumour site, the bacteria then either proliferate in the extracellular space or invade the tumour cells. in either situation, selective cytotoxicity was engineered by expressing toxins, cytokines, tumour antigens, pro-apoptotic factors or prodrugconverting enzymes . non-invasive e. coli has successfully been programmed to invade cultured tumour cells in a hypoxia-responsive or population-density-dependent manner. the corresponding circuitries consist of the anaerobically induced formate dehydrogenase promoter driving the yersinia pseudotuberculosis invasin gene (inv), which mediates invasion using specific integrin receptors that are typically expressed on tumour cells. populationdensity-dependent invasion requires an engineered quorum-sensing circuit that triggers inv expression after the bacterial population has reached a threshold size at the tumour site. this circuitry consists of quorum-sensing receptor luxr that co-induces luxi (which encodes the enzyme producing the quorum-sensing messenger autoinducer (ai- )), and inv. ai- -triggered, luxrmediated expression of luxi represents a positive feedback loop that amplifies inv expression and ai- production; this coordinates and broadcasts the invasion order across the entire population (fig. a) . tumour-invading bacteria have also been engineered for trigger-inducible drug expression after entering tumour cells. in addition to l-arabinose- and γ-irradiation-induced drug expression, a synthetic salicylate-triggered expression device has been used to control expression of drug components following systemic administration of the trigger molecule in mice in tumour cells that have been invaded by salmonella spp. . the device is based on a circuit that is derived from pseudomonas putida, which controls expression of cytosine deaminase in a salicylate-inducible manner . mammalian cells normally lack cytosine deaminase, which means that they are resistant to -fluorocytosine because this enzyme is needed to convert -fluorocytosine into the cytotoxic molecule -fluorouracil. tumour-bearing mice were injected with attenuated salmonella enterica engineered with the p. putidaderived circuit and then treated with -fluorocytosine. the mice showed significant tumour regression when fed with acetylsalicylic acid (aspirin) , which is rapidly converted to salicylate after intake by the animal (fig. b) . rnai is a potent and highly conserved mechanism for the targeted knockdown of mrna translation by small rnas. non-pathogenic e. coli was engineered to express a short rna hairpin that triggers rnai against catenin β- , which is a colon-cancer-specific oncogene . these bacteria, which were also engineered to express proteins to mediate cellular invasion and escape α α from the phagosome, were administered orally or intravenously and significantly reduced catenin β- levels in the intestinal epithelium and in human colon cancer xenografts in mice (fig. c) . combining various bacterial anti-cancer treatment strategies may increase safety, specificity and efficiency in future clinical trials. viral synthetic devices. viruses have also been successfully engineered to transduce specific cells by expressing epitopes that are recognized by particular cell-surface receptors and to express prodrug convertases and cytokines for use in cancer therapy . most of these oncolytic viruses carry coding viral nucleic acids, which may cause side effects owing to recombination with the host chromosome or proviral elements that are already in the host cell. recently, synthetic viral particles have been designed that lack coding nucleic acids and that exclusively package therapeutic proteins, which can be released in a dose-dependent manner . for example, viral particles carrying linamarase from manihot esculenta were injected into human breast cancer xenografts in mice that had been treated with the non-toxic natural product linamarin; these viruses triggered efficient tumour regression owing to the cyanide produced by linamarase-mediated conversion of linamarin (fig. d) . similarly, protein-carrying viral nanoparticles have been used to deliver site-specific dna recombinases, such as flp, to precisely integrate or excise genetic components on the host chromosome . they might also be used to deliver native or chimeric transcription factors that could transiently control the expression of target genes that are involved in therapeutic interventions, lineage control or induction of pluripotency . a transformation sensor for cancer therapy. gene therapy advances for cancer include virus-mediated delivery of cytotoxic effector genes controlled by cancer-specific promoters , or delivery of chimeric adaptor proteins to link tyrosine kinase signalling to the apoptosis-inducing caspase machinery . most promoters and control circuits that coordinate simple reactions such as these are inherently noisy and only allow linear responses, which means limited control of specificity and efficacy. however, using two internal input signals can improve fidelity, mediate sharp response profiles and ensure robust biochemical processes . using decision-making circuits as blueprints, nissim and bar-ziv designed a tunable dual promoter integrator (dpi) to target cancer cells precisely. the dpi consists of two native promoters that are concurrently activated by two independent transcription factors. each cancer-sensing promoter produces a different fusion protein in proportion to its activity, and these two proteins assemble together as a chimeric transcription factor. this transcription factor then activates a synthetic promoter that controls expression of the herpes simplex virus type thymidine kinase (tk ), which is cytotoxic in the presence of nucleotide analogues, such as ganciclovir (fig. a) . the dpi could be optimized for a specific cancer cell type by using different combinations of input promoters and effector genes, as well as by modulating the assembly efficiency and half life figure | synthetic genetic cancer classifiers. a | a transformation-sensing cancer kill switch can consist of a two-input, transformation-sensing device with 'and' logic. the device constantly monitors the transformation state of a cell and produces a kill signal when two malignancy markers occur. two independent malignancy-sensitive promoters drive expression of two chimeric proteins (docs-vp and gal bd -coh ). when they are simultaneously expressed, both proteins dimerize to form a synthetic transcription factor that binds gal operator sites (o gal ), induces downstream minimal promoters (p min ) and triggers expression of the herpes simplex virus type thymidine kinase (tk ). in the presence of ganciclovir, the system is cytotoxic. b | a microrna (mirna)-based cancer classifier that discriminates cancer cells from non-transformed cells by scoring high and low expression profiles of a set of cancer-specific mirnas. the classifier consists of high and low mirna sensors that exclusively promote output gene expression if the specific input mirnas are expressed at high or low levels, respectively. in the high mirna sensor, high-target mirna concentrations prevent translation of mrnas encoding the reverse tetracycline-dependent transactivator (rtta) and the repressor of the lactose operon (laci). this results in derepression of transcription of the output gene (labelled 'output' in the figure). in the low mirna sensor, the output-gene-encoding mrna is only translated when low-target mirna concentrations are present. c | by combining different high and low mirna sensors, the classifier can be customized to sense predetermined profiles of high and low mirna levels, such as the ones that are typically produced by cancer cells and respond with expression of the apoptosis-inducing human bcl -associated x protein (bax). pa, poly(a) tail. a vitamin-a-dependent, g-protein-coupled receptor that is expressed in intrinsically photosensitive retinal ganglion cells networks that replace existing cellular functionality that is ill-driven or out of order. they represent molecular prostheses for non-functional cellular activity; they differ from other synthetic networks that add useful functionality but do not replace non-functional cellular networks. of the chimeric transactivator components. so far, a set of three promoters have been characterized in detail, but the dpi design may accommodate other suitable promoters. the recently developed 'cell-type classifier' is conceptually similar to the dpi, as it can also be programmed to destroy cells that express a specific set of neoplastic markers . the cell-type classifier combines transcription and translation control components in a single scalable synthetic circuit that senses expression levels of a set of (currently up to six) endogenous micrornas (mirnas); it triggers an apoptosis-inducing response only if those levels match a preset profile. the cell-type classifier combines sensor modules for the detection of highly and lowly expressed mirnas (fig. b) . for clinical implementation, both the dpi and the cell-type classifier must either be delivered to the cancer tissue, or they must provide a fail-safe mechanism that constantly eliminates transforming cells from engineered tissue implants. other emerging tools for biomedicine novel treatment strategies will require new technologies to sense and control disease. synthetic biologists have designed new devices that could sense key physiological activities and have found new ways to dose therapeutic interventions precisely in response to external physical cues. such synthetic devices could have wide-ranging biomedical applications. thus far, synthetic control devices that are designed to interface with host metabolism and to reprogram cellular behaviour have largely been limited to heterologous transcription factors. rna controllers may represent an alternative. they are straightforward to design and can be integrated into a single expression unit containing sensors (aptamers), gene-regulatory components (ribozymes) and effector transgenes , , . the inherent modularity and compatibility of rna-based control components enables them to be independently optimized or exchanged. for example, an rna control device consisting of a drug-responsive aptamer linked to a ribozyme in the ′ untranslated region (utr) of a cytokine expression unit enabled trigger-inducible inactivation of ribozymemediated transcript cleavage and full transgene expression in the presence of the input signal . this synthetic rna control device was applied to control proliferation of engineered primary human t cells and enabled external control of the expansion of transgenic t cells that are implanted into mice. synthetic rna control devices could provide the advance that is necessary to enable t cell therapy ; by contrast, state-of-the-art, triggerinducible expansion of engineered t cells using chimeric antigen receptors has only led to moderate proliferation and poor survival of t cells in clinical trials , . another use for synthetic rna is the design of programmable sensor-actuator devices that convert levels of an intracellular protein into a discrete high or low transgene-expression state . the rna devices consist of a three-exon, two-intron minigene followed by the transgene. the introns contain protein-sensing aptamers, and the central exon includes a stop codon. binding of the protein to the aptamers controls splicing of the minigene; when the central exon is spliced out, the transgene is expressed at high levels, and when it remains unspliced, the transgene is expressed at low levels. such a device was configured to sense subunits of nuclear factor kappa b (nfκb) or β-catenin (which are neoplastic markers) and to express the herpes simplex virus thymidine kinase. thymidine kinase renders cells susceptible to ganciclovir, so this device only operated as a cancer kill switch in the presence of the cancer markers and gangcicolvir . the modular configuration of the rna sensor-actuator device allows it to be tailored to different intracellular proteins and even to multi-protein input using specific intronic aptamers. also, responsiveness and performance can be tuned by placing the aptamers at different locations within the introns. the availability of compact rna sensor-actuators that are easy to design and to alter and that control transgene expression in response to intracellular levels of key proteins may also improve the ability to link metabolic disease states with gene-based therapeutic interventions. light is becoming increasingly popular as a traceless, moleculefree input signal for triggering transgene expression in living systems. bacteria have been engineered to record projected images with gigapixel resolution [ ] [ ] [ ] and to adjust transgene expression in response to multichromatic input , and now genetic light switches have also been designed to control gene expression and shape of mammalian cells . devices that convert light pulses into transcription may foster novel therapeutic opportunities in future gene-and cell-based therapies and may improve the manufacturing of difficult-to-produce protein pharmaceuticals, such as cancer therapeutics. an illustrative example is light-controlled expression of the glucagon-like peptide (glp ), which is a promising drug candidate for the treatment of type diabetes (fig. a) . an optogenetic device that enables light-triggered gene expression in human cells was designed. this involves ectopic expression of melanopsin in human embryonic kidney cells and functional rewiring of signalling downstream of melanopsin; the cascade integrates blue-light-pulsetriggered photoreception and produces a reversible and sustained intensity-dependent transcription response. when placed in hollow fibre containers and implanted into mice, transgene expression in the engineered lightsensitive cells could be controlled remotely by an optical fibre . illuminating mice that carried subcutaneous implants of microencapsulated photo-responsive cells also enabled transdermal control of transgene expression and of corresponding protein levels in the blood of treated animals. this system was able to attenuate glycaemic excursions and to control glucose homeostasis in a mouse model of human type diabetes . prosthetic networks. prosthetic networks are synthetic sensor-effector devices that act as molecular prostheses. when engineered into cells and functionally connected to host metabolism, they sense, monitor and score disease-relevant metabolites, process off-level concentrations and coordinate adjusted diagnostic, preventive or therapeutic responses in a seamless, automatic and self-sufficient manner. an example of the use of a prosthetic network is the sensing of metabolites to improve control of urate homeostasis (fig. b) . moderate levels of uric acid, which scavenges radicals, are deemed to be beneficial. however, a transient surge in uric acid that is released by dying cells during cancer therapy leads to tumour lysis syndrome, and chronic hyperuricaemia can result in gout. humans are particularly sensitive to imbalances of urate homeostasis because they lack uricolytic activity. a prosthetic network that constantly monitors blood urate concentrations and restores urate homeostasis by controlled expression of a urate oxidase -which reduces excessive urate concentration while preserving levels that are suitable for radical scavenging -could represent a treatment strategy for hyperuricaemic disorders . in brief, human cells that contain such a prosthetic network have recently been designed by combining: the uric acid sensor hucr , which manages oxidative stress protection in deinococcus radiodurans; the human uric acid transporter urat (also known as slc a ), which increases the intracellular uric acid levels and thus the sensitivity of the prosthetic circuit; and a secretionengineered urate oxidase (smuox) that is clinically licensed for the treatment of the tumour lysis syndrome . figure | advanced therapeutic and prosthetic networks. a | light-triggered transcription control of blood glucose homeostasis. the synthetic phototransduction cascade consists of rewired melanopsin and nuclear factor of activated t cells (nfat) control circuits. photo-isomerization of the -cis-retinal chromophore (r) by blue light (~ nm) activates melanopsin. this sequentially turns on gaq-type g protein (gaq), phospholipase c (plc) and phosphokinase c (pkc) and triggers ca + ion influx via transient receptor potential channels (trpcs) and possibly also from the endoplasmic reticulum. this ca + ion surge activates calmodulin (cam) to calcineurin (can), which dephosphorylates nfat. nfat then translocates into the nucleus, where it binds to specific promoters (p nfat ) and coordinates transgene transcription. when linked to the glucagon-like peptide (glp ), this mechanism allowed light-controlled blood glucose homeostasis to be achieved in a mouse model of type diabetes. b | prosthetic network for the treatment of tumour lysis syndrome and gout. implanted sensor-effector cells are used to monitor serum urate levels constantly: they import urate via a transgenic human uric acid transporter (urat ). urate prevents binding of the uric acid-sensitive transsilencer (krab-hucr, which is the uricase regulator linked to a krab domain) to its operator (huco ). this operator controls expression of secretion-engineered urate oxidase (smuox), so smuox is expressed when urate concentration reaches pathological levels. smuox mediates conversion of urate into allantoin. expression of smuox stops when urate concentration reaches oxidative-stress-protective urate levels. pa, poly(a) tail. part a is modified, with permission, from ref. © ( ) american academy for the advancement of science. the prosthetic uric-acid-responsive expression network (urex) was able to sense uric acid concentrations precisely and to activate secretion of smuox when the uric acid concentration was at pathologic levels. secretion of smuox is stopped as soon as the uric acid concentration has returned to the homeostasis level. this was impressively demonstrated when urex-transgenic cells were implanted into urate-oxidase-deficient mice, which develop acute hyperuricaemia with symptoms that are similar to human gout. urex was able to degrade urate and restore urate homeostasis in the blood, resulting in the dissolution of uric acid crystal deposits in the kidney of treated animals . its straightforward design may allow urex to serve as a blueprint for the assembly of other prosthetic networks that sense metabolic disturbances and circulating pathologic metabolites. an artificial insemination device. artificial insemination is standard practice to facilitate both human reproduction and livestock breeding. because of broad variations in oestrus expression and ovulation timing, coordinating sperm delivery with female oestrus is still a major challenge. ovulation is triggered at a specific time when the pituitary gland releases luteinizing hormone, which binds to the luteinizing hormone receptor (lhr) and coordinates the release of the oocyte. by integrating synthetic signalling cascades with advanced biomaterials, kemmer and colleagues designed an artificial insemination device that coordinates sperm delivery with oestrus control. the artificial insemination device consists of cellulose sulphate capsules containing bull sperm and sensor cells , , . the sensor cells are engineered to express lhr constitutively so that when it is activated, it triggers expression of cellulase that can be secreted. after implantation into the cow's uterus, the sensor cell line constantly monitors the animal's luteinizing hormone levels, and the oestrus-triggered surge in luteinizing hormone levels leads to the production of secreted cellulase, which degrades the implanted capsule and results in the timely delivery of the sperm and successful conception. fine tuning of the designer cascade could enable its use in other species, including in humans. in recent years, synthetic biology has substantially advanced strategies for classical biomedical applications, such as pathogen characterization , , , disease analysis [ ] [ ] [ ] , diagnostics [ ] [ ] [ ] , screening assays , , , , , drug production [ ] [ ] [ ] [ ] and vaccination , , . this progress may imminently develop into shorter drug discovery , and drug development timelines, increased precision of drug delivery , and production of new and more affordable medicines [ ] [ ] [ ] [ ] [ ] [ ] . ultimately, sophisticated therapeutic sensor-effector devices that can sense disturbances, seek out pathological conditions and restore function are on the roadmap. such therapeutic networks that connect diagnostic input with therapeutic output may provide all-in-one diagnostic, preventive and therapeutic solutions in future gene-and cell-based therapies. matching diagnostic outcome with high-end therapies has recently become a focus of the pharmaceutical industry, which has declared personalized medicine as the treatment strategy of the future. tools that will have a tremendous impact in future biomedical applications include: using light-activated triggers to bring about a precise therapeutic response in cells , programming bacteria to seek and destroy cancer cells , , and using synthetic circuitry to keep crucial metabolites at homeostatic levels , , to manage disease-controlled expansion or to eliminate specific cell populations , , . recent work has shown that this is, in principle, possible and that some devices are working as expected and are producing a therapeutic impact in animal models of human diseases , . implants consisting of engineered microencapsulated cells represent a way of introducing prosthetic networks with a predefined function instead of directly targeting the host cells with the genetic material. although implants containing cells with engineered prosthetic networks are certainly the most promising way forward, they will limit biomedical applications to extracellular disease metabolites that can be therapeutically addressed through the vascular system. however, there is still a long way to go until syntheticbiology-based biomedical devices will be a clinical reality. placing therapeutic circuits in specific cells of a patient and making sure that there will be no interference with human metabolism are the most important challenges. therefore, clinical use of synthetic-biology-based devices and therapeutic scenarios will face the same scientific, ethical and legal issues as any gene-and cell-based therapy, but they may offer more complex control dynamics and are therefore expected to have a higher therapeutic impact. although none of the synthetic devices, prosthetic networks and related products that are pioneered by synthetic biologists have yet been used in the clinics or in clinical trials, the stage is set -with novel treatment strategies available and the commitment of the pharmaceutical industry in place -for synthetic biology to deliver the biomedicines of the twenty-first century. network news: innovations in st century systems biology evolvability and hierarchy in rewired bacterial gene networks identification of functional elements and regulatory circuits by drosophila modencode informing biological design by integration of systems and synthetic biology genome-sequencing anniversary. a celebration of the genome, part i genotype and snp calling from next-generation sequencing data scalable gene synthesis by selective amplification of dna pools from high-fidelity microchips high-fidelity gene synthesis by retrieval of sequence-verified dna identified using high-throughput pyrosequencing parallel on-chip gene synthesis and application to optimization of protein expression creating bacterial strains from genomes that have been cloned and engineered in yeast evidence for large diversity in the human transcriptome created by alu rna editing hematopoietic stem cell gene therapy with a lentiviral vector in x-linked adrenoleukodystrophy creation of a bacterial cell controlled by a chemically synthesized genome life after the synthetic cell making cellular memories a synchronized quorum of genetic clocks diversity-based, model-guided construction of synthetic gene networks with predicted functions a synthetic oscillatory network of transcriptional regulators a synthetic gene-metabolic oscillator construction of a genetic toggle switch in escherichia coli an engineered mammalian band-pass network hysteresis in a synthetic mammalian gene network an engineered epigenetic transgene switch in mammalian cells rationally designed logic integration of regulatory signals in mammalian cells a universal rnai-based logic evaluator that operates in mammalian cells a fast, robust and tunable synthetic gene oscillator intron length increases oscillatory periods of gene expression in animal cells a tunable synthetic mammalian oscillator a synthetic-natural hybrid oscillator in human cells a synthetic time-delay circuit in mammalian cells and mice multi-input rnai-based logic circuit for identification of specific cancer cells this paper describes a synthetic network that classifies cells according to their specific microrna expression profiles. this strategy might be generally applicable for identification andengineering of specific cell types synthetic biology: integrated gene circuits mammalian synthetic biology-from tools to therapies recent advances in mammalian synthetic biology -design of synthetic transgene control networks synthetic gene networks in mammalian cells molecular diversity-the toolbox for synthetic gene switches and networks rna-based computation in live cells ligand-dependent regulatory rna parts for synthetic biology in eukaryotes frameworks for programming biological function through rna parts and devices refinement and standardization of synthetic biological parts and devices mammalian synthetic biology: engineering of sophisticated gene networks ultrasensitivity and noise propagation in a synthetic transcriptional cascade semi-synthetic mammalian gene regulatory networks intronically encoded sirnas improve dynamic range of mammalian gene regulation systems and toggle switch origin of bistability underlying mammalian cell cycle entry synthetic ecosystems based on airborne inter-and intrakingdom communication streptogramin-based gene regulation systems for mammalian cells controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin de novo design and construction of an inducible gene expression system in mammalian cells development of genetic circuitry exhibiting toggle switch or oscillatory behavior in escherichia coli multistability in the lactose utilization network of escherichia coli a genetic time-delay circuitry in mammalian cells engineered bidirectional communication mediates a consensus in a microbial biofilm consortium programmed population control by cell-cell communication and regulated killing a synthetic multicellular system for programmed pattern formation watch the clockengineering biological systems to be on time a synthetic low-frequency mammalian oscillator synthetic biology: exploring and exploiting genetic modularity through the design of novel biological networks eukaryotic systems broaden the scope of synthetic biology synthetic biology: applications come of age next-generation synthetic gene networks the second wave of synthetic biology: from modules to systems a designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms this describes the first synthetic closed-loop control gene network that manages homeostasis of a crucial disease metabolite in an animal model the first optogenetic device that controls the production of a therapeutic protein in an animal disease model is the potential of synthetic biology: a view from the european academies science advisory council synthetic biology moving into the clinic characterization of the reconstructed spanish influenza pandemic virus complete chemical synthesis, assembly, and cloning of a mycoplasma genitalium genome a two-amino acid change in the hemagglutinin of the influenza virus abolishes transmission infectious diseases. as swine flu circles globe, scientists grapple with basic questions origins and evolutionary genomics of the swine-origin h n influenza a epidemic synthetic viruses: a new opportunity to understand and prevent viral disease synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice antibody-profiling technologies for studying humoral responses to infectious agents anti-borrelia burgdorferi antibody profile in post-lyme disease syndrome lips arrays for simultaneous detection of antibodies against partial and whole proteomes of hcv, hiv and ebv mutations of the igbeta gene cause agammaglobulinemia in man oligomeric organization of the b-cell antigen receptor on resting cells autoantigen discovery with a synthetic human peptidome this study is a demonstration of how the synthesis of the human peptidome enables the direct identification of new autoantigens a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection this provides a description of a standard strategy for the production of codon-pair deoptimized viral genomes for use as attenuated life vaccines optimization and quantification of protein synthesis inside liposomes from pond scum to pharmacy shelf heat-stable oral alga-based vaccine protects mice from staphylococcus aureus infection female-specific insect lethality engineered using alternative splicing female-specific flightless phenotype for mosquito control this paper presents a strategy in which transgenic insects containing a synthetic gene network could provide pest control by disseminating a conditional flightless female phenotype among natural insect populations genetic elimination of dengue vector mosquitoes a synthetic homing endonuclease-based gene drive system in the human malaria mosquito science snipes at oxitec transgenicmosquito trial macrolide-based transgene control in mammalian cells and mice design of a novel mammalian screening system for the detection of bioavailable, non-cytotoxic streptogramin antibiotics the impact of synthetic biology on drug discovery in this study, the reconstruction of a synthetic antibiotic resistance network in mammalian cells enabled the discovery of novel anti stringent rosiglitazone-dependent gene switch in muscle cells without effect on myogenic differentiation a system for small-molecule control of conditionally replication-competent adenoviral vectors streptomyces-derived quorumsensing systems engineered for adjustable transgene expression in mammalian cells and mice synthetic ethr inhibitors boost antituberculous activity of ethionamide controlled proliferation by multigene metabolic engineering enhances the productivity of chinese hamster ovary cells in vitro assays for anticancer drug discovery-a novel approach based on engineered mammalian cell lines a novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells drug discovery and natural products: end of an era or an endless frontier? exploiting plug-and-play synthetic biology for drug discovery and production in microorganisms chemical biology: synthetic metabolism goes green silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture this work described the biosynthesis of halogenated molecules in plants using synthetic pathways production of the antimalarial drug precursor artemisinic acid in engineered yeast isoprenoid pathway optimization for taxol precursor overproduction in escherichia coli engineering escherichia coli for production of functionalized terpenoids using plant p s the impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies conditional dna-protein interactions confer stimulus-sensing properties to biohybrid materials drug-sensing hydrogels for the inducible release of biopharmaceuticals genetically engineered protein in hydrogels tailors stimuli-responsive characteristics synthetic mammalian gene networks as a blueprint for the design of interactive biohybrid materials a gene therapy technology-based hydrogel for the trigger inducible release of bipharmaceuticals in mice a coumermycin/novobiocin-regulated gene expression system regulation of protein secretion through controlled aggregation in the endoplasmic reticulum this paper describes a novel metabolite-based strategy for the eradication of antibiotic-tolerant bacterial 'persister cells bacterial charity work leads to population-wide resistance dispersing biofilms with engineered enzymatic bacteriophage sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis how antibiotics kill bacteria: from targets to networks a common mechanism of cellular death induced by bactericidal antibiotics engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy fighting bacterial infections-future treatment options engineered bacterial communication prevents vibrio cholerae virulence in an infant mouse model secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes engineering the perfect (bacterial) cancer therapy environmentally controlled invasion of cancer cells by engineered bacteria genetically engineered salmonella typhimurium as an imageable therapeutic probe for cancer tumour-targeted delivery of trail using salmonella typhimurium enhances breast cancer survival in mice in vivo gene regulation in salmonella spp. by a salicylate-dependent control circuit short hairpin rna-expressing bacteria elicit rna interference in mammals reprogrammed viruses as cancer therapeutics: targeted, armed and shielded therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles protein transduction from retroviral gag precursors efficient construction of sequencespecific tal effectors for modulating mammalian transcription purification and characterization of -aminocyclopropane- -carboxylate oxidase from apple fruit targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis redirecting tyrosine kinase signaling to an apoptotic caspase pathway through chimeric adaptor proteins how erk / activation controls cell proliferation and cell death: is subcellular localization the answer? a tunable dual-promoter integrator for targeting of cancer cells engineering ligandresponsive gene-control elements: lessons learned from natural riboswitches artificial ribozyme switches containing natural riboswitch aptamer domains genetic control of mammalian t-cell proliferation with synthetic rna regulatory systems control of large, established tumor xenografts with genetically retargeted human t cells containing cd and cd domains the promise and potential pitfalls of chimeric antigen receptors in this paper, the abundance of specific cellular proteins was linked to transcription of target genes via splicing-modulating rna controllers. this enables proteome-based classification and engineering of individual cells synthetic biology: engineering escherichia coli to see light multichromatic control of gene expression in escherichia coli a synthetic genetic edge detection program induction of protein-protein interactions in live cells using light spatiotemporal control of cell signalling using a light-switchable protein interaction hucr, a novel uric acid-responsive member of the marr family of transcriptional regulators from deinococcus radiodurans cloning and expression in escherichia coli of the gene encoding aspergillus flavus urate oxidase a novel system for trigger-controlled drug release from polymer capsules design of high-throughputcompatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa short-term bmp- expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells an autonomous system for identifying and governing a cell's state in yeast the authors declare no competing financial interests. key: cord- - nb z authors: oh, hyang soon; uhm, dong choon title: current status of infection prevention and control programs for emergency medical personnel in the republic of korea date: - - journal: j prev med public health doi: . /jpmph. . sha: doc_id: cord_uid: nb z objectives: emergency medical personnel (emps) are pre-hospital emergency responders who are at risk of exposure to infections and may also serve as a source for the transmission of infections. however, few studies of infection control have specifically addressed emps in the republic of korea (hereafter korea). the goal of this study was to assess the current status of infection prevention and control programs (ipcps) for emps in korea. methods: a cross-sectional survey was conducted to quantitatively assess the resources and activities of ipcps. a total of emps in five metropolitan cities completed a structured questionnaire from september to january . the data were analyzed using descriptive statistics, multi-response analysis, and the chi-square test. results: the mean age of the participants was . ± . years. ipcps were found to have weaknesses with regard to the following resources: the assignment of infection control personnel (icp) ( . %), hand hygiene resources such as waterless antiseptics ( . %), the use of paper towels ( . %), personal protective equipment such as face shields ( . %), and safety containers for sharps and a separated space for the disposal of infectious waste ( . %). likewise, the following activities were found to be inadequately incorporated into the workflow of emps: education about infection control ( . %), post-exposure management ( . %), and the decontamination of items and spaces after use ( . %). icp were found to have a significant effect on the resources and activities of ipcps (p< . ). the resources and activities of ipcps were found to be significantly different among the five cities (p< . ). conclusions: ipcps for emps showed some limitations in their resources and activities. ipcps should be actively supported, and specific ipcp activities for emps should be developed. during the middle east respiratory syndrome (mers) outbreak in the republic of korea (hereafter korea) from may to july , two emergency medical personnel were infected with mers, among a total of healthcare workers who developed mers occupationally [ ] . emergency medical personnel are also vulnerable to other infections when they provide care, including blood-borne pathogens [ , ] , hospital pathogens such as methicillin-resistant staphylococcus aureus [ , ] , and highly contagious epi-infection control for emergency medical personnel demic diseases such as severe acute respiratory syndrome [ ] , swine-origin influenza a (h n ) [ ] , and ebola virus [ ] . in addition, inadequately trained rescue personnel may serve as a source of disease transmission between hospitals and the community [ ] . therefore, infection prevention and control programs (ipcps) for emergency medical personnel are important both to protect them and to prevent the spread of transmissible diseases and pathogens to patients, other healthcare workers, and the general population. although some studies have addressed infection control among emergency responders globally, this issue has not been adequately studied in the korea. the sole study addressing this issue was published in and assessed bacterial contamination in ambulances and other environments that emergency medical workers commonly come into contact with [ ] . in , ipcps with standard operating procedures for emergency medical personnel were first established [ ] . in , the act on rescue and emergency services specified the first ipcp-based regulations for emergency medical personnel. article regulated infection control and article regulated the cleaning, disinfection, and sterilization of ambulances and equipment [ ] . finally, in , ipcps for emergency medical personnel were first implemented in accordance with those regulations. however, no survey addressing the basic patterns of infection control among emergency medical personnel has been conducted. therefore, the aim of this study was to evaluate the current status of ipcps for emergency medical personnel, including the availability of infection control resources (i.e., human resources and infrastructure) and the provision of infection control activities (i.e., employee health, surveillance, education, and decontamination) in order to provide basic data about ip-cps for emergency medical personnel. a cross-sectional survey was conducted in five representative large metropolitan cities in the korea. this study used a self-reported anonymous questionnaire. completion of the survey took approximately minutes. the questionnaire was a modified version of that used by oh et al. [ ] , which was originally developed based on the study on the efficacy of nosocomial infection control (senic) [ ] . the senic instrument was modified for this study based on regulations [ , ] for ipcps for emergency medical personnel (i.e., the enforcement of rescue and emergency service regulations for emergency medical personal and emergency activities), with the goal of thoroughly identifying and measuring the characteristics of the actual ipcps employed by emergency medical personnel during their work duties. we also sought to increase the validity of the questionnaire to the greatest extent possible. the questionnaire was organized into three parts assessing the general characteristics, resources, and activities of ipcps for emergency personnel according to the senic methodology [ ] [ ] [ ] . part a included questions assessing participants' demographic characteristics and the general conditions of their work places, which were mostly fire stations. these questions addressed issues including types of decontamination procedures and the presence of separate spaces for infectious waste disposal. part b included questions assessing the resources of the ipcps, including human resources and infrastructure for implementing ipcps, the assignment of part-time infection control personnel (icp), the assignment of part-time staff for decontamination, the frequency of infection control committee meetings, infection control guidelines, and hand hygiene equipment (hand antiseptics and hand drying methods) both in the station and in in the ambulance, personal protective equipment (ppe), the types of gloves and safety containers for the prevention of sharps injuries, and decontamination procedures. part c included questions assessing the following activities of ipcps: employee health programs; post-exposure management programs; vaccination programs; health screening programs; surveillance programs for patients, emergency medical personnel, and spaces and instruments; education; and decontamination activities. the questionnaire was revised following the administration of a pilot version to five expert emergency medical personnel. the final questionnaire included questions. part c (activities) was found to have good internal consistency, with a cronbach's alpha of . . parts b (resources) and a (general characteristics) had relatively good internal consistency, with cronbach's alpha values of . and . , respectively. using a convenience sampling method, first responders were recruited from the five largest cities in korea (referred to as a through e). this sample comprised approximately % of the total emergency medical personnel from these metropolitan cities. a power analysis using g* power version . . . was. the sample size required for a statistical power of . , an effect size of . , and an alpha < . was n = . a total of questionnaires were distributed via mail, and three reminder follow-up calls were made to increase the response rate. ultimately, questionnaires (response rate, . %) were collected from september through january . the study was approved by the institutional review board (irb) of daejeon university (irb no. - -hr- - ). the investigators first contacted the emergency rescue service directors in each of the five cities to obtain permission to recruit participants. questionnaire packages were distributed with a return envelope. in order to foster confidentiality, participants were not required to complete the survey at work. participation was both voluntary and anonymous. the kolmogorov-smirnov test was used to analyze data distribution and normality. descriptive statistics were calculated, including percent distributions for general characteristics and questions pertaining to infection control resources and infection control activities. multiple-response questions were also analyzed. categorical variables were compared using the chisquare test to identify significant differences in ipcps among emergency personnel. we used column-rate comparison in the chi-square test, which involved comparing the ratios across columns for each response, in order to identify significantly different columns for a given row. variables that did not meet normality assumptions were analyzed using the non-parametric one-sample chi-square test. significant differences are noted in the tables using superscripts in the american psychological association format. a p-value < . was considered to indicate statistical significance. positive answers about variables assessing resources and activities were scored as , while negative answers were scored as . multi-response questions were scored based on the number of responses selected. however, three questions were scored differently, as described below. a question about the annual number of infection control committee meetings was scored as for responses indicating that such meetings were held twice or more annually, while other answers were scored as , because it has been recommended that such meetings be held at least twice annually [ , ] . another question regarding hand-drying methods was scored as if paper towels were used, while the other answers were scored as , because paper towels have been recommended as the proper hand-drying method in the healthcare area [ ] . another question about the frequencies of cleaning or disinfection of ambulance surfaces and items that have been used was scored as for answers indicating that this was performed weekly, while all other answers were scored as , because it has been recommended that these procedures be carried out at least weekly [ , ] . after scoring the variables, stepwise multiple regression analysis with a forward selection procedure was used to study the variables that may have affected the resources and activities of ipcps. the t-test was used to analyze differences in resources and activities depending on the presence of icp. analysis of variance (anova) and the post-hoc test using the scheffé method were used to analyze differences in resources and activities among in the metropolitan cities. all statistical analyses were performed using spss version . (ibm corp., armonk, ny, usa). a p-value < . was considered to indicate statistical significance. the general characteristics of participants and their working conditions are presented in table . the majority of respondents were males between and years of age (mean age, . years). the participants' primary job titles were emergency medical technician (emt) paramedics ( . %), basic emts ( . %), and nurses ( . %). most of the participants ( . %) were college or university graduates. the majority had worked as emts for fewer than five years ( . %). the participants were mostly firefighters who worked a three-shift schedule ( . %). the resources available for ipcps are illustrated in table . emt paramedics ( . %) made up the majority of icp personnel and they were most often assigned the responsibility for decontamination ( . %). infection control committees generally met twice per year ( . %) or at least three times per year ( . %). of the respondents, . % indicated that infection control guidelines had been developed for their work environments. waterless hand antiseptics were used most frequently ( . %). paper towels ( . %) and cotton towels ( . %) were used as hand-drying methods with approximately equal frequency. hand antiseptic was not universally present ( . %), and face shields were infrequently available in ambulances ( . %). latex ( . %) and vinyl gloves ( . %) were equally available in ambulances. safety containers for sharps were generally available in ambulances ( . %). autoclaves were the most common method used for sterilization ( . %), followed by ultraviolet sterilizers ( . %), automatic cleaners ( . %), ultrasonic cleaners ( . %), and other methods ( . %). a separate space for the disposal of infectious waste was infrequently available ( . %). infection control activities are presented in table . a small proportion of participants ( . %) participated in post-exposure management programs. influenza vaccination ( . %) was the most frequently reported vaccination, followed by hepatitis b ( . %) and tetanus and diphtheria ( . %). most respondents reported two health screenings per year ( . %). the majority of respondents described surveillance programs of the workplace environment ( . %), emergency medical personnel ( . %), and patients ( . %). education about infection control was reported by . % of participants. most respondents ( . %) indicated that ambulance surfaces and used items were decontaminated weekly. a decontamination checklist was used by . % of participants. the results of the cross analysis are presented in table . a comparison of variables according to the presence of icp and infection control resources showed that the presence of decontamination personnel (p< . ), infection control committees (p= . ), infection control guidelines (p< . ), and hand antiseptics in the ambulance (p = . ) were significantly higher when icp were assigned than when icp were not assigned. in addition, vaccinations (p< . ), health screen- multiple-response questions. ings (p= . ), and surveillance (p= . ), were significantly more frequent when icp were present. when variables were compared according to location, significant differences were found among the five cities included in our study, with the exception of infection control committees and surveillance ( table ). the presence of icp and the assignment of personnel for decontamination were significantly higher in city c than in other cities (p< . ). the development of infection control guidelines was significantly more common in cities a and c than in other cities (p< . ). hand antiseptics and hand antiseptics in ambulances were significantly more common in cities a, c, and e (p< . ). among infection control activities, vaccination was significantly higher in city c (p= . ). health screenings were significantly lower in city c (p< . ). education about infection control was significantly higher in city a (p< . ). decontamination of ambulances and used equipment was significantly less frequent in cities a and c (p< . ). values are presented as number (%). superscripts are presented using the american psychological association format, in which different superscripts within a row indicate statistically significant differences ( a, b means statistically different subgroups). *p< . . the results of the multiple regression analysis are presented in supplemental table . the regression model for resources showed good fit, with r-squared and adjusted r-squared val- ues of . and . , respectively. the durbin-watson statistic was . in a model summary, and the regression model showed a value of p< . in a one-way anova table. the regression model for activities likewise showed good fit, with r- squared and adjusted r-squared values of . and . , respectively. the durbin-watson statistic was . in a model summary, and the p-value of the regression model was < . in a one-way anova table, indicating good fit. however, the low r-squared value indicates that these models could explain only a small amount of the variation in the dependent variables. cities, sex, educational background, work experiences, and the presence of icp showed a significant association with ipcp resources. cities and icp presence also showed a significant association with ipcp activities, and the presence of icp had a greater impact than all other variables on both the resources and activities. the mean scores of total resources and activities were significantly higher when icp were present than when no icp were present. the mean scores of total resources and activities were significantly different among cities (supplemental table ). the mean scores of resources and activities were comparable in cities a, b, and c. however, the mean score for resources in city d was relatively lower than other cities, although the mean score for activities in city d was higher. contrastingly, the mean score for resources in city e was relatively high, while the city e displayed the lowest mean activity score (supplemental figure ). this is the first study to provide an overview of the current status of basic ipcps among emergency medical personnel in the korea. the general characteristics of the participants were representative of the general characteristics of korean emergency medical personnel. the emergency medical personnel sampled were highly educated and relatively young. if these professionals are educated and well trained through repeated and systematic infection control programs, ipcps for emergency medical personnel can be expected to improve despite the weaknesses identified in this study. the basic ipcp resources for emergency medical personnel identified in this study did not fully satisfy pre-existing recommendations, with the exception of the frequency of meetings of infection control committees [ , ] . in , the rescue and emergency services regulations recommended that every fire station be assigned part-time icp, develop and implement infection control guidelines, and ensure that infection control committees meet twice a year [ , ] , because these elements were identified as essential prerequisites of ipcps [ , ] . hand hygiene is the most basic and effective method for infection control [ ] . however, the use of hand antiseptics and paper towels showed severe deficiencies in comparison to what is prescribed by regulations and recommendations [ , , ] . the frequent use of cotton towels has the serious potential problem of hand recontamination. therefore, hand hygiene resources, such as hand antiseptics and disposable paper towels, should be supplied in quantities sufficient to satisfy the guidelines. the provision of ppe in the ambulance should also follow guidelines in order to prevent occupational exposure to blood and other bodily fluids [ , , ] . gloves are the most basic ppe used for the prevention of occupational exposure to blood or bodily fluids. however, since vinyl is an inadequate material for protecting the skin from infectious materials, gloves should be changed to materials that are impermeable and stronger. safety containers for the disposal of sharps and facial masks should also be included in all ambulances, as they are essential items for occupational safety and protection from infectious materials [ ] . many different types of cleansers, disinfectants, and sterilizers were used. in order to ensure that such equipment is used for decontamination in the most effective possible way, the personnel responsible for decontamination should be specially trained in both the operation of the specific equipment in question and in risk-stratification principles in decontamination [ ] . fire station facilities for emergency medical personnel need separate spaces for the disposal of items after use, which our data show are currently lacking. thus, further assessments and improvements in decontamination procedures should be required. deficiencies were shown in infection control activities, including post-exposure management programs, surveillance, education, the decontamination of ambulance surfaces and equipment, and the use of decontamination checklists. health screening programs were the only area that satisfied regulations [ , ] . post-exposure management programs were found to be especially uncommon. post-exposure programs should be developed to minimize the impact of needle stick injuries and other routes of exposure to blood-borne pathogens [ , , ] . surveillance and education programs should be established with greater frequency. the weekly decontamination of ambulance surfaces and equipment and the use of a checklist for decontamination are areas for improvement in order to satisfy the relevant regulations [ , ] . icp were found to be a significant factor affecting ipcps in pre-hospital settings among emergency medical personnel and paramedics [ , ] . when icp were assigned, the availability of resources and the extent of infection control activities were significantly higher. the resources available to ipcps and the activities of ipcps varied significantly among the five cities included in this study. we found that the metropolitan city d had a significantly lower proportion of resources in terms of icp, decontamination personnel, infection control guidelines, and hand antiseptics. however, this city had a significantly higher proportion of activities (e.g., health screenings and decontamination processes), and respondents from this city showed a significantly higher score for activities than for resources. in contrast, the metropolitan city e had a significantly higher proportion of resources in terms of decontamination personnel, infection control committees, and hand antiseptics. however, this city also had a significantly lower proportion of activities (e.g., vaccination, and surveillance), and respondents from this city reported the lowest mean activity scores. further evaluation is needed in all metropolitan cities, and particularly in the metropolitan city e, in order to evaluate the effectiveness of ipcps. in order to maintain consistent ipcps, standardized nationwide programs should be developed and implemented. this study had some limitations in that the study sample was not fully representative of the national population, because we selected representatives from five metropolitan cities in the republic of korea, and the distribution of respondents among the cities was unequal. in spite of the limitations of this study, our findings provide a baseline regarding ipcps for emergency medical personnel, which is important because emergency medical personnel have been neglected in national infection control systems and academic research in this area. emergency medical personnel should be included in nationwide infection control systems because they are vulnerable to occupational infections, such as mers or other infections [ , , , , ] . in conclusion, ipcps for emergency medical personnel showed weaknesses in human resources (e.g., the, assignment of icp in general and for decontamination in particular), infrastructure (e.g., the use of paper towels, ppe, and safety containers for pre-vention of sharps injuries), and activities (e.g., education and decontamination). icp were also identified as a factor that had a significant effect on ipcps for emergency medical personnel. the availability of ipcp resources and activities differed by location. icp assignment, the use of disposable paper towels, ppe, and safety containers, as well as the consistent implementation, distribution, and development of ipcp resources should be put into practice both in all five of the large metropolitan cities included in this study and nationwide in the korea. middle east respiratory syndrome information: mers daily report hepatitis c virus infection among firefighters, emergency medical technicians, and paramedics: selected locations safeguarding our first responders: infection control and prevention for firefighters exposure of emergency medical responders to methicillin-resistant staphylococcus aureus methicillin resistant staphylococcus aureus in ambulances hospital infectious disease emergency preparedness: a survey of infection control professionals interim guidance infection control for emergency medical personnel for emergency medical services (ems) systems and - - public safety answering points (psaps) for management of patients with confirmed or suspected swine-origin influenza a (h n ) infection nosocomial and community-acquired infection rates of patients treated by prehospital advanced life support compared with other admitted patients risk stratification-based surveillance of bacterial contamination in metropolitan ambulances daejeon fire department. rescuer safety management sop. daejoon: daejeon fire department ministry of government legislation. revised decree about rescue and first-aid development and application of evaluation indices for hospital infection surveillance and control programs in the republic of korea the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals staffing and structure of infection prevention and control programs world health organization. who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care guideline for isolation precautions: preventing transmission of infectious agents in health care settings guideline for disinfection and sterilization in healthcare facilities guideline for infection control in health care personnel occupational exposures in emergency medical service providers and knowledge of and compliance with universal precautions we wish to thank the subjects who participated in this study. the authors have no conflicts of interest associated with the material presented in this paper. key: cord- -wh s ws authors: gao, da-peng; huang, nan-jing title: optimal control analysis of a tuberculosis model()() date: - - journal: appl math model doi: . /j.apm. . . sha: doc_id: cord_uid: wh s ws in this paper, we extend the model of liu and zhang (math comput model : - , ) by incorporating three control terms and apply optimal control theory to the resulting model. optimal control strategies are proposed to minimize both the disease burden and the intervention cost. we prove the existence and uniqueness of optimal control paths and obtain these optimal paths analytically using pontryagin’s maximum principle. we analyse our results numerically to compare various strategies of proposed controls. it is observed that implementation of three controls is most effective and less expensive among all the strategies. thus, we conclude that in order to reduce tuberculosis threat all the three controls must be taken into consideration concurrently. tuberculosis (tb) is a major global health threat caused by mycobacterium tuberculosis (mtb) that most often affects the lungs (pulmonary tb) but can affect other sites as well (extrapulmonary tb). fortunately, tb is a preventable and curable disease. following the world health organization (who), more than million patients have been successfully treated in countries that adopted the who strategy since [ ] . however, the global burden of tb remains enormous. in , there were an estimated . million new cases of tb ( % co-infected with hiv) and . million people died from tb [ ] . in , there were million new tb cases and . million tb deaths (in which . million are hiv-positive patients) [ ] . the increase of new cases lies in multiple factors such as the spread of hiv, the collapse of public health programs, the emergence of drug-resistant strains of mtb [ ] [ ] [ ] and exogenous re-infections, where a latently-infected individual acquires a new infection from another infectious individual (see, for example, [ , ] and references therein). the tb control approach is the chemoprophylaxis treatment in the absence of an effective vaccine. neglecting the compliance with drug treatments might result in a relapse and antibiotic resistant tb, i.e. multidrug-resistant tb (mdr-tb) [ ] . mdr-tb is one of the most serious health problems and the progress in curing that is slow. critical funding gaps for tb care and control make it difficult to sustain recent gains, promote further research and develop new drugs and vaccines [ ] . it is well known that the mathematical models play vital roles in the dynamics and control of many epidemics including malaria, severe acute respiratory syndromes (sars) and tb (see, for example, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and references therein). many mathematical dynamic models for tb have been studied extensively in the literature; for instance we refer the reader to [ ] [ ] [ ] [ ] [ ] [ ] . "fast progressors" and "slow progressors" are two different ways considered by most models to progress to active disease after infection. the probability of latent and treated individuals who might be reinfected [ ] is also taken into account by some latest models as the infection and/or disease do not confer full protection which is already well recognized [ ] . some previous models of tb, especially the predictive models endeavor to calculate a threshold called basic reproductive number. the dynamics of transmission of the disease has been analyzed in terms of the reduction of the basic reproductive number. since the first paper published by jung et al. [ ] in , the time dependent optimal control strategies have been employed in the study of dynamics of tb mathematical models by many authors (see, for example, [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). valuable theoretical results generated by both approaches of studying control strategies can be used to guide epidemic control programs. various objective criteria may be adopted, according to a chosen goal (or goals). in , considering that the treated individuals are also infectious, liu and zhang [ ] proposed a nonlinear tb model system as follows: where a population of size n ( t ) is partitioned into five subclasses: susceptible ( s ( t )), vaccinated ( v ( t )), individuals infected with tb in latent stage ( l ( t )), individuals infected with tb in the active stage ( i ( t )) and treated individuals infected with tb ( t ( t )). the parameter represents the constant recruitment rate of susceptible individuals. parameter p is the rate at which susceptible individuals are moved into the vaccination process. the natural death rate is μ. the disease-induced death rate coefficient for individuals in compartment i is α. parameter ρ represents the rate that individuals successfully treated of tb return to the latent tb stage and parameter γ is the treatment rate in the infective class. it is assumed that the time before latent individuals become infectious obeys an exponential distribution, with mean waiting time δ . thus, δ is the rate at which an individual leaves the latent compartment to become infectious. susceptible individuals acquire tb infection from individuals with active tb at rate βs(i + ρ t ) , where β is the disease transmission coefficient, and the parameter ρ < accounts for the reduction in infectiousness among individuals with active tb who are treated (in comparison to those who are not treated). fraction l of susceptible individuals who acquire tb infection is assumed to enter the latent tb class ( l ), at the rate βs(i + ρ t ) , while the remainder moves to the active tb class ( i ). it is assumed that individuals in the latent class do not transmit infection. the vaccinated individuals are infected at rate ρ βv (i + ρ t ) , where ρ < represents the reduction in risk of infection owing to vaccination (for a vaccine that offers % protection, ρ = ; thus in reality < ρ < ). for model ( . ) , liu and zhang [ ] first obtained the basic reproduction number . after that they showed that if the basic reproduction number r of the model system is below one then disease dies out otherwise it persists. this indicates that the basic reproduction number, r , plays a crucial role in characterizing dynamics of the disease and could be used to suggest or design tb control strategies. however, they did not further investigate time dependent control strategies since their discussions had been concentrated on prevalence of tb at equilibria. note that, ∂ p | ( . ) < , also due to liu and zhang [ ] , which implies that increasing the vaccination rate of susceptible individuals against infection of tb is conducive to disease control. in order to understand the dynamics under what circumstances tb can be controlled or curtailed, we implement the theory of optimal control. three intervention strategies, called controls, are included in model system ( . ) . we write controls as functions of time and assign reasonable upper and lower bounds to them. first, a "case finding" (identification of latently infected individuals) control mechanism is incorporated in model ( . ) by replacing constant vaccination rate with u ( t ). second, a "case holding" (treatment of individuals with active tb) control mechanism is incorporated in model ( . ) by replacing constant successfully treatment rate with u ( t ). finally, another "case holding" is instituted in model ( . ) by replacing constant treatment rate with u ( t ). it is required that u ( t ), u ( t ) and u ( t ) are bounded lebesgues integrable functions. we intend to determine optimal control strategies that minimize not only the infected individuals but also the cost of these three controls. as mentioned by kumar and srivastava [ ] , the larger proportion of population is covered, the higher efforts and expenditure is required to provide vaccination. hence, it is suitable to use the nonlinearity of order four to describe the high expensiveness concerned with the vaccination process. following the idea of kumar and srivastava [ ] , in this paper, the cost constructions that account for high nonlinear relationship (nonlinearity of order four i.e. u (t) ) between cost and effort s during vaccination process has been employed in the application of optimal control to tb mathematical models. using the same parameters and class names as in model system ( . ) , the system of differential equations describing the controlled model is as follows: ( . ) in view of the fact that vaccination and treatment are easily available and implementable control strategies to stop tb, we obtain model system ( . ) by considering vaccination and treatment as control variables from model system ( . ) . the detailed analysis of optimal control problem is given in the following sections. the subsequent part of this paper is as follows: we present the formulation of the optimal control problems in section . in section , we investigate the existence of an optimal control function and derive an optimal system characterizing the optimal control. in section , we analyze the uniqueness of the optimal system, in which the state system coupled with co-states. it is worth pointing out that the proof of existence and uniqueness involving biquadratic form of the control variables has not been explored so far as we know. we perform numerical simulations to illustrate our theoretical results in section . the paper ends with a conclusion in section . this section is devoted to the study of our model system ( . ) , when we administered vaccination and treatment policies over a fixed time window. for that we design an objective functional by keeping the biomedical goal in our mind. our goal is to minimize the number of infected individuals (including latent, infectious, being treated individuals) with tb virus while at the same time keeping the cost of implementing these three control strategies very low. in mathematical perspective, for a fixed terminal time t f , the problem is to minimize the objective functional here, the total cost on a finite time horizon [ , t f ] consists of the cost induced by the disease itself and the cost induced by vaccination and treatment effort s. we split the disease cost into the cost induced by latent group, proportional to the number of individuals infected with tb in latent stage, the cost induced by infected group, proportional to the number of individuals infected with tb in the active stage, and the cost induced by treated group, proportional to the number of treated individuals infected with tb. modeling the control cost is a more delicate issue. the cost involved in vaccination process requires relatively higher efforts and expenditure because of covering larger proportion of population. for example, vaccination efforts target first quarter of population will be less than the next quarter and so on. therefore, the cost for covering larger proportion of population involved in vaccination process will be not only increasing but also growth of cost will be faster and steeper. hence, we consider the biquadratic form in the vaccination control to represent high expensiveness of vaccination process [ ] . that is, the cost involved in vaccination process is taken as it is also assumed that cost of treatment is nonlinear and takes a quadratic nature, which is found to be consistent with previous works in the literature (see, e.g. [ ] ). in other words, the cost incurred in improving successfully treatment rate (the effort s such as regularity of drug intake) is taken as similarly, the cost incurred in providing treatment is taken as ) are the weight constants of the infected tb individuals and control measures. they can be chosen to balance cost factors due to size and importance of the parts of the objective functional. we assume that there are practical limitations on the maximum rate at which individuals may be vaccinated or treated during a given period of time and the maximum successfully treated rate. in the rest of this paper, for simplicity of notation, we represent u (t) = u , u (t) = u and u (t) = u . we seek optimal controls u , u and u in u ad such that is the control set. in optimal control theory, there are several basic problems included such as to prove the existence of an optimal control, to characterize the optimal control, to prove the uniqueness of the control, to compute the optimal control numerically and to investigate how the optimal control depends on various parameters in the model. in this section, we will study the sufficient condition for the existence of an optimal control of our system of ordinary differential equations ( . ) . we refer to the conditions in theorem iii . and its corresponding corollary in [ ] . after that, we characterize the optimal control functions by using pontryagin's maximum principle [ , ] , and then we derive necessary conditions for our control problem. in this subsection, we will investigate the existence of an optimal control of our model ( . ) . the boundedness of solutions to system ( . ) for finite time interval is needed to establish the existence of an optimal control and the proof of the uniqueness of the optimality system. we begin by examining the priori boundedness of the state solutions of model ( . ) . for model system ( . ) to be epidemiologically meaningful, it is important to prove that all its state variables are nonnegative for all time. in other words, solutions of model system ( . ) with nonnegative initial data remain nonnegative for all time t > . suppose, for example, the variable s becomes zero for some time to establish the upper bounds for the solutions, we consider an equation for the total population size n . the rate of change of the total population, obtained by adding all the equations in model ( . ) , is given by proof. to prove this theorem, we follow the requirements from theorem . and corollary . in [ ] and verify nontrivial requirements. let r(t, x , u ) be the right-hand side of ( . ) . we need to show the following conditions are satisfied: ( ) r is of class c and there exists a constant c such that ( ) the admissible set f of all solutions to system ( . ) with corresponding control in u ad is nonempty; in order to verify above conditions, we write then it is easy to see that r(t, x , u ) is of class c and | r(t, , ) | = . moreover, one has since s, v, l, i and t are bounded, there exists a constant c such that this means that condition ( ) holds. thanks to condition ( ), there exists a unique solution to system ( . ) for a constant control, which further implies that condition ( ) holds. in addition, condition ( ) is obvious from the definition and the proof of this theorem can be completed by verifying condition ( ) . in order to show the convexity of the integrand in the objective functional g(t, x , u ) , we have to prove that and so the proof is complete. since there exists an optimal control for minimizing function ( . ) subject to system ( . ) , we drive the necessary conditions for this optimal control by using pontryagin's maximum principle [ , ] . we need to define hamiltonian for deriving the necessary conditions as, here, λ = (λ , λ , λ , λ , λ ) ∈ r is known as adjoint variable. the optimality system of equations is found by taking the appropriate partial derivatives of hamiltonian ( . ) with respect to the associated state variables. the following theorem is a consequence of the maximum principle. given an optimal control pair (u * , u * , u * ) and corresponding solutions to the state system s * , v * , l * , i * , t * , that minimize objective functional ( . ) , there exists adjoint variables λ , λ , λ , λ and λ , satisfying , u max , proof. the result follows from a direct application of a version of pontryagin's maximum principle for bounded controls [ , ] . the differential equations governing the adjoint variables are obtained by differentiation of hamiltonian function ( . ) , evaluated at the optimal control. then the adjoint system can be written as follows: ( . ) and = , , ) . we point out that the optimality system consists of the state system ( . ) with the initial conditions, adjoint system ( . ) with transversality conditions ( . ) , and optimality condition ( . ) . thus, we have the following optimality system: due to the boundedness for the state system, the adjoint system has bounded coefficients which is linear in each of the adjoint variables. therefore, the adjoint system have finite upper bounds. we will state the following proposition (without proof) needed for the proof of the uniqueness of the optimality system for the small time window. proposition . . [ ] the function u * (m ) = min { max { m, a } , b} which is lipschitz continuous in m, with a < b for some fixed nonnegative constants. now, we are in position to prove the uniqueness of the optimality system in the same way as shown in [ , , ] . proof. the proof is done by contradiction. note that the boundedness for the state system and the adjoint system, the lipschitz continuity of optimal control functions, and some elementary inequalities play key roles in obtaining a contradiction. , u max ; for ease of notation, we generally omit the dependence on time in the following except in the case that a specific time is intended. we consider the difference of the resulting equations for x i and x i , y i and y i , i = , , . . . , . from the first equation of ( . ) , we get dx dt and d x dt it follows from the above two equations that ( . ) multiplying both sides of ( . ) by (x − x ) and then integrating both sides from to t f , one has in order to simplify the right-hand expressions of ( . ) , we need some elementary inequalities. an elementary inequality it follows that | a − b| ≤ (a + b ) and thus | a by the elementary inequality (a + b) ≤ (a + b ) , we have where c depends on bounds for x , y . another inequality is also needed to obtain the specific estimate: where m i (i = , , . . . , ) and n j ( j = , , . . . , ) depend on the coefficients and the bounds of the state variables and co-state variables. table . table . we employed parameters values similar to those reported in previous studies whenever possible. table provides a summary of the parameter values used in numerical simulation of model ( . ) . due to lack of data, we make hypothesis on some parameter values within realistic ranges. the initial values for the states are given by table . table . table . table . we start comparing the case of the population with optimal control actions and without control actions. in figs. - , the population with optimal control actions are shown in solid lines, compared with the population without control actions which are shown in dashed lines. we observe from fig. that the susceptible population is higher under control compared to the case without control. the results in fig. predict a higher level of vaccinated population for the aforementioned tuberculosis intervention strategies under control compared to the case without control. the results depicted in figs. and clearly suggest that the optimal control results are very effective in control of individuals infected with tb in latent stage and in the active stage, as expected. in fig. , the population of treated humans declines sharply in the first years and after that decrease further, while fig. shows slight increase in the last two years of the intervention strategy. this is due to the fact that the treatment control u becomes zero after years. the simulation results in fig. suggest that this strategy would require that the vaccination control u should be at maximum for almost the entire period of intervention, while treatment controls u and u should maintain maximum effort s f or years and years respectively before decreasing to zero. in what follows, we consider different control strategies in the reduction of infected individuals (including latent, infectious, being treated individuals). we observe in fig. that the infected individuals are lowest when three controls are considered. as the cost is one of the important parameter to decide for applicable strategy, we performed the cost design analysis and made comparative study of the cost for various strategies. the cost profiles for various control strategies are given in fig. . note that the cost is least when three controls are considered, that is, the implementation of three control measures to restrict the epidemic while at the same time keeping the cost of vaccination and treatment very low are good policies for the achievement of our goal. in fig. we present the effect of varying the disease transmission coefficient β on the number of individuals infected with tb in the active stage ( i ( t )). we observe that after about years the effect of β is negligible on individuals infected with tb in the active stage. fig. shows the effect of varying fraction coefficient l on the number of individuals infected with tb in the active stage ( i ( t )). it is easy to see that the individuals infected with tb in the active stage affected much by l . in this work, we have studied a simple mathematical model of tb presented by liu and zhang [ ] . in order to control the spread of tb, we applied a preventive control in the form of vaccination and two treatment controls to susceptible, latent, and individuals infected with tb in the active stage. next, we established the objective functional by remembering the fundamental biomedical goals in our mind. theoretically, we proved the existence of optimal control and studied the characterization of optimal control by pontryagin's maximum principle. in order to assure that the optimal control is unique in the depictions, we investigated the uniqueness of the optimal control system. numerically, the simulations of the extended tb model ( . ) indicate that the vaccination and treatment strategies are effective in reducing tb disease transmission, especially we may get maximum disease control by using minimum cost as shown in figs. and . it could therefore be concluded that the comprehensive effect of all the three controls not only minimizes cost burden but also keeps a tab on infective population. undoubtedly, dynamics of tb infection is far more complicated and varied than the one captured by this mathematical model. therefore, we will incorporate the time delay for drug resistance into the present model so as to provide us a better assessment for designing tb dynamics. in addition, we note that the principle technique for optimal control problem is to solve a set of "necessary conditions" that an optimal control and corresponding state must satisfy. if the hamiltonian is linear in the control variable u , it would be difficult to solve for u * from the optimality equation. we will leave these cases as our future work. tuberculosis control: past years and future progress evolution of who, - 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(x − x ) + (y − y ) + (y − y ) .(iv) case of x (y − y ) < x ( y − y ) < . by aid of an easy induction gives thatwhere k ≥ + d . in order to simplify the right-hand of ( . ) , another common expression can be used repeatedly,where c depends on bounds for x and y . based on the above arguments, we findwhere m , n are appropriate upper-bounds. similarly, we can obtain the following inequalities for (x i (t f ) , x i (t f )) and (y j ( ) , y j ( )) with i = , , , and j = , , , , :now it follows from ( . ), ( . ), ( . ), ( . ), ( . ), ( . ), ( . ), ( . ), ( . ), ( . ) that (x − x ) dt will be nonnegative. similar arguments apply to remaining integral terms, we can obtain all of the other λ s and t f s . take the maximum of all of the λ s used as λ and the minimum of the t f s used as t f , then the coefficient of each integral term in ( . ) is nonnegative. this implies that hence the solution of ( . ) is unique for small time. this ends the proof. remark . . we note that theorem . shows that the solution of ( . ) is unique when t f is small. as pointed out by lenhart and workman [ ] , we can usually obtain uniqueness of the solutions for ( . ) , but only for small time t f . this small time condition is due to reverse time orientations of the state equation and adjoint equation. in this section, a fourth-order runge-kutta algorithm is used to solve optimal system ( . ) numerically. with initial conditions for the states, chosen an initial guess for the controls, the state differential equations ( . ) are solved forward in time by a fourth-order runge-kutta scheme. with the application of the current iteration solution of the state equations ( . ) and the transversality conditions ( . ) , the adjoint system ( . ) is solved backward in time, again, by a fourth-order runge-kutta scheme. both state and adjoint values are used to update the controls with the characterizations given by ( . ) , and then the iterative process is repeated. this procedure is continued iteratively till current state, adjoint, and control values converge sufficiently. key: cord- - ibeidyw authors: goldrick, barbara a. title: the practice of infection control and applied epidemiology: a historical perspective date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: ibeidyw the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved since the landmark study on the efficacy of nosocomial infection control project, and infection control professionals (icps) must continue to develop the knowledge and skills necessary to practice infection prevention and control. practice analyses of infection control conducted between and were analyzed to determine changes in practice. these data reflect a % increase in infection control activities over a -year period. however, resources for infection control and prevention have not kept pace with this increased activity. in addition, the current trend toward mandatory reporting of health care-associated infections (hais) among several states will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the association of professionals in infection control and epidemiology, inc, continues to explore the issue of mandatory reporting of hais. the practice of infection control and applied epidemiology: a historical perspective barbara a. goldrick, rn, phd, mph, cic chatham, massachusetts the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved since the landmark study on the efficacy of nosocomial infection control project, and infection control professionals (icps) must continue to develop the knowledge and skills necessary to practice infection prevention and control. practice analyses of infection control conducted between and were analyzed to determine changes in practice. these data reflect a % increase in infection control activities over a -year period. however, resources for infection control and prevention have not kept pace with this increased activity. in addition, the current trend toward mandatory reporting of health care-associated infections (hais) among several states will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the association of professionals in infection control and epidemiology, inc, continues to explore the issue of mandatory reporting of hais. (am j infect control ; : - .) the major infection control movement emerged in the united states in the s. and by the mid- s, thousands of hospitals throughout the country had infection surveillance and control programs (iscps) in place. however, it was not until the landmark study on the efficacy of nosocomial infection control (senic), which was conducted by the centers for disease control (cdc) in the mid- s, that the link between iscps and the reduction of nosocomial infections (nis) in acute care facilities was established. the senic demonstrated that effective iscps were associated with a % reduction in nis. the senic project found that, prior to , very few hospitals in the united states had an infection control professional (icp), but, by , % of the hospitals had at least icp. in the early s, the cdc recommended that hospitals have at least full-time equivalent (fte) icp for every beds ; however, the senic found that only % of the icps surveyed worked full-time specifically on infection control activities, and the amount of time was related to hospital size, with an overall average of hours per week. the extensive revisions to the joint commission on accreditation of hospitals (jcah) guidelines for infection control, which formally recommended a position responsible for surveillance, added further impetus to include icp positions to hospital infection control programs. until the senic, there were no published studies that assessed the dimensions of infection control practice; therefore, the senic project included a study of the role of the icp. nearly all ( %) of icps surveyed in - were registered nurses (rns), with two thirds of these having a diploma in nursing, one fourth a baccalaureate degree, and % an associate degree. the senic also identified practice activities among icps: surveillance, policy development, training, epidemic investigations, and consulting (table ) . however, there were no certification standards for the icp at the time of the senic project. the association for practitioners in infection control, inc. (apic), a multidisciplinary organization, was established in to meet the education and practice needs of icps in the united states. the name was changed to the association for professionals in infection control and epidemiology, inc, in to recognize the organization's maturation and evolution into the broader context of health care delivery in the united states. apic's sister organization, community and hospital infection control association-canada (chica-canada), was incorporated in for icps practicing in canada. the apic developed educational standards for infection control practice in , which included epidemiology; microbiology; infectious diseases; sterilization, disinfection, and sanitation; patient care practices; education; management and communication; and employee health. these standards were consolidated into the apic curriculum for infection control practice, which was published in . the first infection control practice analysis (pa) was conducted in at the request of the certification board of infection control (cbic), which was established in . the first pa survey collected demographic data and data from a task inventory rating scale, which consisted of task statements, categorized according to the educational standards of apic. a randomized stratified sample of icps who would receive the pa questionnaire was deliberately skewed toward larger hospitals (. beds) because the pa committee felt that these hospitals would most likely employ icps who performed the full range of infection control activities. based on inclusion/exclusion criteria, a total of icp respondents' data were used for analysis to determine key tasks for infection control practice. most ( %) of respondents in the pa survey were rns who worked in community hospitals ( %) with . beds ( %). all but % of the respondents' hospitals were accredited by the jcah. although the majority of the respondents were full-time employees, % spent less than hours/week in infection control, and many held multiple positions within the hospital; however, % of the icps surveyed indicated that infection control was their primary responsibility. the majority ( %) of respondents had been in infection control practice between and years, and nearly all ( %) had attended educational programs in infection control. sixty tasks (activities) related to the areas/dimensions of infection control practice were identified as relevant in the pa (table ). the pa was the basis for the first infection control certification examination, which was offered in the united states in . it also defined and described the scope of infection control practice for the first time and established a baseline for measuring progress and changes in practice. recertification for icps is required every years; therefore, the cbic repeats the pa every years. by the s, infection control practice had expanded beyond the acute care setting. infection control practice also had changed considerably. new infectious diseases such as acquired immunodeficiency syndrome had emerged, prospective payment systems for hospitals were in place, and the joint commission on accreditation of health care organizations (jcaho; formerly jcah) instituted outcome measures as part of the accreditation process. these changes required new or enhanced skills for the icp. therefore, in , years after the first infection control pa, a study was conducted to update and revalidate the original ( ) pa to ensure that the content of the certification examination was a valid representation of infection control practice. a modified delphi technique was used between panels of icps and further validated by a third panel of subject matter experts for a total of icps. there was a high level of agreement between respondents on the pa, the icps, and the expert panel, providing content validity for the certification examination based on current infection control practice. eight new tasks (activities) were identified for a total of tasks in areas (dimensions) of infection control practice (table ) . these additional tasks ''reflect[ed] an expanding role of the icp as a planner and manager.'' the cbic conducted its second pa survey among icps in , which included a random sample of canadian icps for the first time. the original pa list was used with modifications to reflect current infection control practice and terminology. a total of responses were available for analysis. the majority ( %) of respondents were rns; % held an associate degree; % held a baccalaureate degree; and % held an advanced degree. the majority of respondents had years or more in infection control practice ( %), worked in hospitals with greater than beds ( %) less than hours a week ( %), and were certified in infection control ( %). ninety-five tasks identified in the pa were organized into major practice dimensions describing the responsibilities of icps in the united states and canada: infectious process, surveillance/epidemiologic investigation, transmission of infection, management and communication, and education (table ) ; however, new tasks were added, and outdated tasks were eliminated. each pa builds on those conducted previously and is an important component of ensuring content validity that reflects current infection control practice. this process of content validation involves systematic collection of information that describes behaviors and activities (tasks) performed by occupants of the practice in question. because of the numerous changes in health care delivery, which affected the scope and practice of infection control since the pa, a more contemporary analysis of infection control practice was needed in the pa survey conducted by the cbic. the application of decision rules ensured that the resulting certification examination accurately reflected infection control practice in the united states and canada, regardless of the region or size of facility. a representative sample across all health care settings was selected from the apic and the chica-canada icps to receive the pa questionnaire. a return of responses significantly exceeded the n required by power analysis. as in past pa surveys, the majority ( %) of respondents in the survey were rns. thirteen percent of respondents held an associate degree; % held a baccalaureate degree; and % had an advanced degree. the majority of respondents in the pa survey worked in acute care settings ( %), worked in facilities with greater than beds ( %), and worked less than hours per week in infection control ( %). nineteen percent of the respondents worked in long-term care ( %), mental health facilities ( %), and rehabilitation centers ( %). thirty-five percent of the respondents had or more years of experience in infection control practice, but less than half ( %) were certified in infection control. the completed inventory on the pa resulted in tasks organized into major practice areas, as listed in table . the apic/chica-canada infection control and epidemiology: professional and practice standards were published in . the document has the following sections: ( ) professional standards that the icp is expected to meet or exceed and ( ) practice standards that the icp is capable of meeting, regardless of applicability to the specific practice setting. these professional and practice standards were synthesized into the most current pa survey, which was conducted by the cbic in , to ensure that the resulting content outline was consistent with current standards of infection control practice. the primary source for the pa survey questionnaire was the infection control professional task list. the target population for the survey consisted of icps in the united states and canada who met the eligibility requirements for taking the certification examination. a final sample of responses were available for analysis in the pa survey, which significantly exceeded the n required by power analysis. therefore, results from this sample could be generalized to icps in the united states and canada. in the continuing process of serving the international infection control community, the survey instrument also was distributed to a selected sample of international icps. table outlines the major areas of infection control practice identified in the pa. two changes in the major areas of practice were made. these included a category that incorporates both education and research. research had not been considered in previous pas; however, because there were only tasks associated with the ''research'' category, it was combined with the ''education'' category, and the category was renamed ''education and research.'' several new tasks associated with employee health also were identified. as a result, all aspects of employee health were separated into a new category: ''infection control aspects of employee health,'' which had previously been included in the category that addressed preventing and controlling the transmission of infectious agents. table indicates that tasks related to the areas of infection control practice were identified in the pa. the majority of respondents in the pa survey was rns ( %), held a baccalaureate degree or advanced degree ( %), and was certified in infection control ( %). more than half ( %) of the respondents had been in infection control practice more than years. most of the respondents ( %) worked in an acute care hospital with $ beds ( %) with # full-time equivalent icp ( %), and nearly half ( %) worked or more hours a week. eight percent of the respondents worked in long-term care (compared with % in ), % worked in public health, and % worked in mental health facilities. the pa reflected changes in the practice of infection prevention/control and applied epidemiology at that time and identified the responsibilities of the icp both in the united states and in canada. however, several new infection control activities have been identified since . to remain current, the cbic will conduct its next pa in , which will include tasks related to changes in infection control practice since . table illustrates that the practice of infection control evolved significantly between and . figure shows the increase in infection control tasks from to between and , a % increase over a -year period, with little or no additional resources allotted to infection control programs during that time. although the pa survey was undertaken early in , long before the september , , attacks on the united states and the department of homeland security was formed, the cdc developed a plan to upgrade the nation's public health infrastructure to respond to acts of biological terrorism. in , the apic and the cdc developed a bioterrorism readiness plan ''. to serve as a tool for [icps] and health care epidemiologists to guide the development of practical and realistic response plans for their institutions in preparation for a real or suspected bioterrorism attack.'' as a result, many icps across the united states developed policies and procedures based on the apic and the cdc recommendations to prepare their health care facilities to deal with bioterrorism, and, although bioterrorism was not a separate infection control practice category listed in the pa, the tasks related to attacks of biologic warfare are another layer in the practice of infection control, which deals with the prevention and control of the transmission of infectious agents (table ). since the september attacks, there has been a substantial increase in icps involvement in emergency preparedness and management for bioterrorist attacks and emerging infections. for example, the recent emergence of severe acute respiratory syndrome (sars) and the threat of a new influenza pandemic are further examples of the need for due diligence in infection control. the main lesson learned from the sars outbreak was that it was contained through the conscientious application of enhanced infection control measures at the national and local levels. these same measures will defeat sars should it reemerge. control of an emerging infection requires swift action by health care providers and an adequate public health infrastructure. as change in the health care system continues, infection control practice also must evolve. icps are instrumental in ensuring that their health care facilities comply with new regulations and guidelines, which include education of health care workers on proper hand hygiene, prevention of needlestick injuries, isolation techniques, and appropriate use of personal protective equipment, and are prepared for emerging and reemerging infectious diseases. in addition, changes in the us health care infrastructure occurred during the past years when many hospitals merged or became part of multihospital health systems that included health care facilities across the continum of care. these changes in the organization and delivery of health care services prompted an expansion of the role of many icps to include not only the responsibility for infection control programs in acute care and/or long-term care facilities but the added responsibility for nonacute health services such as freestanding surgery centers, medical and dental clinics, rehabilitation units, child and adult day care centers, and home care. however, icps report that resources for the infection control staff have remained static despite the need to respond to emerging infections and implementation of new regulations and guidelines. emergence of multidrug-resistant microorganisms in all health care facilities also has necessitated increased icp activity. o'boyle et al used a delphi method to study infection control tasks in addition to those found in the pa, along with additional responsibilities. a panel of experts (n ) from states, who represented icps in acute care, long-term care, and community settings, identified new tasks added to the listed in the pa. the delphi panel also estimated the percentage of time used in the major infection control practice domains. the activity with the greatest average estimated time was surveillance ( %), followed by education ( %), prevention ( %), and communication ( %). the activity with the least average estimated time was control measures ( %). lack of adequate resources was seen as influencing icps' ability to perform tasks across all infection control functions. the panelists in the study determined that a ratio of . to . icp for every occupied acute care beds was adequate infection control staffing. despite the additional responsibilities involved in the practice of infection control, the ratio of icp for every acute care beds has continued in many health care facilities in the united states since the senic project. however, the ratio of icps per beds was a recommendation that was not evidence based. two recent reports recognized that the complexity of the current practices of icps has changed considerably since the senic and that the old ratio of icp per beds is no longer adequate. the authors also recommend that infection control programs be based on the scope of the infection control program rather than bed size. , in the past year alone, new guidelines for infection control practice were published. icps must be aware of these guidelines and implement those recommendations that are strongly evidence based. the cdc recently released draft documents of revised isolation procedures and tuberculosis prevention guidelines. when the final documents are published, icps must implement the new recommendations. in addition, revised jcaho infection control standards, which went into effect in january , include an emergency management plan (standard ic. . ) that requires health care facilities to respond to epidemics and infections that may require expansion of patient care over extended periods of time. the first report, which addressed the ratio of icps per number of beds since the senic project, was published in the canadian journal of infection control in the summer of . a canadian infection control alliance, consisting of infection control experts, was asked to reach consensus on the key components and resources needed to support effective infection prevention and control programs across the health care continuum: acute care settings, long-term care facilities, and community and home care settings. the alliance recommended full-time equivalent icps for every beds in acute care settings and full-time equivalent icp for every to beds in long-term care facilities. the projected needs for icps in each of these settings was determined based on the expertise represented by members of the alliance. and the canadian infection control alliance study were conducted before the september attacks on the united states and therefore do not reflect current infection control practice, which now includes emerging infections and bioterrorism preparedness. one state, new jersey, recently recognized the importance of infection control programs and published revised hospital licensure regulations that mandate an icp ratio of full-time icp per adjusted-occupied beds as a minimum standard. the adjusted-occupied bed calculation takes into account patient days, outpatient factors, and case mix. also mandated was certification by the certification board in infection control and epidemiology for all icps within years of beginning infection control practice. the landmark institute of medicine (iom) report on medical errors identified nosocomial infection surveillance as a model for voluntary patient safety reporting systems. the national nosocomial infection surveillance (nnis) system, created by the cdc in to establish a national nosocomial infections database, is the nation's largest and oldest performance measurement system devoted to hospital-acquired infections. the nnis system started with hospitals in , and, by , approximately hospitals were participating in the nnis system. participation in the nnis system is voluntary and involves only acute care facilities in the united states. surveillance data are collected uniformly by trained icps using standardized protocols that target inpatients at high risk of infection and are reported routinely to the cdc at which they are aggregated into a national database. participating hospitals are assured by law that the cdc will not provide any information that would identify any individual or institution and that the data received will be held in strict confidence. icps in the nnis system collect data for selected ''surveillance components'': adult and pediatric intensive care units, high-risk nursery, and surgical patients, using standard cdc definitions that include both clinical and laboratory criteria. nnis data provide benchmarks to guide hospitals within and outside the nnis system to improve efforts aimed at reducing infection rates. nnis reports are published in the biomedical literature on a regular basis, with the latest providing data from january through june . the infrastructure of the nnis system offers a national resource on which to build improved voluntary patient safety monitoring efforts, as outlined in the iom report. other than the nnis system, there currently is no national standardized method for collecting hospital infection data. in addition, because each hospital monitors those infections and procedures that are most risky for their specific patient populations, all hospitals do not monitor the same infections. nonetheless, by early , states-pennsylvania, missouri, illinois, virginia, and florida-had new regulations, which mandate that icps report hais to state agencies; however, each state's requirements differ. for example, the missouri regulations call for the ''methodologies and systems for data collection established by the federal centers for disease control and prevention national nosocomial infection surveillance system, or its successor .'' the florida bill, on the other hand, would allow patients to request and obtain information about hospital infection rates. mandatory reporting of hais is currently pending in at least other states. legislation for mandatory reporting of hais in california was vetoed by governor schwarzenegger, making the following case: ''. infection control programs have considerable merit and are currently in effect. the department of health services and the joint commission on accreditation of health care organizations scrutinize hospital infection control programs and the national quality initiative is expected to more than double the number of quality indicators tracked by may . this calls into question the need of a new program to address this issue .. the absence of data auditing and review by impartial clinical experts may call into question the quality and ultimate validity of the data on hospital-acquired infections ..'' the healthcare infection control practices advisory committee (hicpac) (formerly the hospital infection control practices advisory committee), which is authorized under the public health service act, advises the secretary, department of health and human services (dhhs) and the cdc regarding the practice of infection control and strategies for surveillance, prevention, and control of hais, antimicrobial resistance, and related events in settings in which health care is provided. the committee also advises the cdc on periodic updating of existing guidelines, development of new guidelines, and other policy statements regarding the prevention of hais. in response to the recent states' legislation regarding mandatory public reporting of hais, hicpac released a document, guidance on public reporting of healthcare-associated infections, which includes recommendations for use by policy makers and organizations that are tasked to design and implement public reporting systems for hais. however, based on an extensive review of the scientific literature, hicpac found no conclusive evidence for or against public reporting of hais as a method to prevent or control their occurrence in health care settings. the apic supports the right of consumers and purchasers of health care to expect quality health care and responsible public reporting of performance indicators. in its position paper entitled ''release of nosocomial infection data,'' the apic outlined specific guidelines for interhospital comparison of hai surveillance data. these included ( ) trained icps who use standardized protocols for data collection, ( ) maintenance of a continued level of surveillance over time, ( ) consistent use of valid case definitions for identifying infections, ( ) appropriate use of denominator data and time periods for rate-based data, and ( ) risk stratification to control for different levels of illness among patients. in keeping with its philosophy, mandatory reporting of hais has become a high priority issue for the association. on march , , the apic released its ''position on mandatory public reporting of healthcare-associated infections.'' the apic continues to explore this issue with other stakeholders and to identify and develop informational resources to assist its members at the local level. the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved, and icps must continue to develop the knowledge and skills necessary to practice infection prevention and control as changes in health care, standards, guidelines, and regulations evolve. practice analyses of infection control conducted between and reflect an increase in infection control activities from to tasks in areas of infection control practice, a % increase over a -year period. however, resources have not kept pace with the increase in infection control activities. in addition, the recent trend toward public reporting of hais will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the apic continues to explore this issue. the emergence of infection surveillance and control programs in us hospitals: an assessment, the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals the infection control nurse in us hospitals, - : characteristics of the position and its occupant a hospital program for control of nosocomial infections joint commission on accreditation of hospitals (jcah) association for professionals in infection control and epidemiology (apic) community and hospital infection control association (chica)-canada educational standards of the association for practitioners in infection control the apic curriculum for infection control practice. volumes i and ii certification board of infection control and epidemiology (cbic) part ii: tasks, knowledge, and abilities for practice a task analysis of infection control practitioners, . part i: methodology and demography validating the certification process for infection control practice job analysis : infection control practitioner practice analysis: building the foundation for validity job analysis : infection control practitioner apic/chica-canada infection control and epidemiology: professional and practice standards practice analysis for infection control and epidemiology in the new millennium biological and chemical terrorism: strategic plan for preparedness and response bioterrorism readiness plan: a template for healthcare facilities public health grand rounds staffing requirements for infection control programs in us health care facilities: delphi project requirements for infrastructure and essential activities of infection control and epidemiology in out-of-hospital settings: a consensus panel report requirements for infrastructure and essential activities of infection control and epidemiology in hospitals: a consensus panel report. society for healthcare epidemiology of america the state of the science of health care epidemiology, infection control, and patient safety critical access hospitals surveillance, prevention and control of infection development of a resource model for infection prevention and control programs (ipcps) in acute, long term, and home care settings: conference proceedings of the infection prevention and control alliance development of a resource model for infection prevention and control programs (ipcps) in acute, long term, and home care settings: conference proceedings of the infection prevention and control alliance canadian report: icp needs across the health care continuum institute of medicine (iom) national nosocomial infections surveillance (nnis) system report, data summary from characteristics of hospitals and infection control professionals participating in the national nosocomial infections surveillance system section consumer union healthcare infection control practice advisory committee (hicpac) healthcare infection control practice advisory committee (hicpac) association for professionals in infection control and epidemiology (apic) the next report from the cdc containing comparative healthcare-associated infection rates, which will include data from the national nosocomial infections surveillance (nnis) system and the first full year of data from the new national healthcare safety network (nhsn), will be published in june . although nnis hospitals began collecting data under the new nhsn protocols beginning in january , they have not been able to report these data to cdc yet, because of unforeseen complexities encountered in launching the web-based reporting tool. until the new report is available, the nnis report issued in october (published in december ) may be used for comparison purposes. key: cord- - ndp ru authors: xu, pengbo; wu, di; chen, yuqin; wang, ziwei; xiao, wei title: the effect of response inhibition training on risky decision-making task performance date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: ndp ru response inhibition is an important component of executive function and plays an indispensable role in decision-making and other advanced cognitive processes. at the same time, we need an effective way to improve decision-making in the face of complex and limited information. this study mainly explored the influence of response inhibition training on college students’ risky decision-making. the recruited students were randomly divided into the training group (n = ) and the control group (n = ). the training group engaged in go/nogo and stop-signal tasks for weeks, while the control group was given the task of reading and summarizing popular science articles related to self-control. the stroop task and balloon analog risk task were used to evaluate the pretest and posttest performance in inhibitory control and risky decision-making tasks, respectively, for all subjects. the results showed that response inhibition training can be effectively transferred to interference control task performance. the results showed that both the reward acquired and adjusted balloon analog risk task score (adj bart) significantly improved compared to the pretest in the training group, while the control group showed no significant differences in the reward acquired and the adj bart between the pretest and the posttest. although response inhibition training increased risky behaviors in the balloon analog risk task, it substantially reduced overly conservative behaviors and participants gained more money. there are all kinds of risky decisions that we make in life. from daily shopping to financial investments, people always need to make choices with limited time and information resources. many studies have shown that people's decision-making is influenced by gender, individual characteristics, emotional states, cognitive abilities, irrelevant information, and so on (sun et al., ; stanovich, ; talukdar et al., ; weller et al., ) . however, little is known about whether a simple and an operational training method can effectively affect decision-making. in the dual-process theories of decision-making, system processes are often automatic, fast, and easily affected by emotion, while system processes are relatively slow and rational process, in which the most important function of system is the successful override of system (stanovich and west, ; evans and stanovich, ; stanovich, ) . moreover, the selection of alternative responses in the decision-making process also depends frontiers in psychology | www.frontiersin.org on the continuous effectiveness of regulatory control processes (moeller et al., ) . that is, to achieve better decision-making, it is necessary to continuously control their own dominant responses and irrelevant interference information, which is undoubtedly closely related to the ability of inhibitory control. the purpose of this paper is to determine whether primary cognitive training can effectively change high-level decisionmaking behavior. if the method of improving risky decisionmaking ability through primary cognitive training (such as inhibitory control) is proven and widely accepted, it will greatly advance the research process in the field of decision-making and will certainly provide a direction for future development. inhibitory control, one of the important components of executive function, is the ability to suppress irrelevant, interfering, incorrect or inappropriate goal-directed dominant responses, impulses, behavioral choices, and automatic behavioral habits (barkley, ; miyake et al., ; munakata et al., ; enge et al., ) . inhibitory control can be roughly divided into reaction inhibition and interference control; the former mainly focuses on the suppression of the dominant response, while the latter focuses on the suppression of irrelevant information. many studies have shown that inhibitory control plays an important role in verbal communication (bishop and norbury, ) , reading comprehension (wang and gathercole, ; potocki et al., ) , memory retrieval (depue et al., ; penolazzi et al., ) , and mathematical ability (gilmore et al., ) and is involved in other higher cognitive processes, such as problem-solving and decision-making (sakagami et al., ; shenoy and yu, ) . in addition, inhibitory control training can affect working memory and fluid intelligence (liu et al., ) , and there have already been some practical applications in controlling addictive behavior, losing weight, reducing diet consumption, and improving mental illness (houben, ; bartholdy et al., ) . therefore, inhibitory control can be considered a basic ability that people must have. the traditional view is that inhibitory control is an internal top-down execution process (aron et al., ). an increasing amount of research results shows that top-down implementation of frontal regions is not always necessary to inhibit control behaviors and the participation of these inhibitory regions can be automatically driven by specific stimuli (lenartowicz et al., ) . manuel et al. compared event-related potentials (erps) before and after auditory go/nogo task training and found decreased activity in the left parietal cortex (manuel et al., ) , suggesting that the repeated and stable association between the stimulus and inhibition response in the go/nogo task resulted in the gradual separation of top-down connectivity in the frontal lobe, thus facilitating rapid automatic inhibition. this is also the purpose of inhibitory control training, that is, training the slow thought suppression process into an automated, faster process. there are many disputes about whether there is a close relationship between performance in the inhibitory control task and the risky decision-making task. kertzman et al. ( ) used the performance on the go/nogo task, the matching familiar figures test (mfft), and the stroop task as indicator of inhibition ability and used the iowa task performance as an indicator of risk and found no direct correlation between the two (kertzman et al., ) . they suggested that although there is some overlap between inhibitory control and the cognitive processing of risky decision-making, they may represent two relatively independent abilities. this may have something to do with the limitations of the iowa gambling task itself. previous studies have demonstrated that short-term stop-signal task (sst) training can reduce risk-taking behavior in gambling tasks (verbruggen et al., ; stevens et al., ) . they designed a special training study and systematically studied the generalization model of promoting automatic inhibition and developing a top-down control inhibition training program. simply training people to control their exercise behavior induced them to make cautious and risk-averse decisions for at least h and the effect was comparable to that found in previous studies that used transcranial direct current stimulation (tdcs) to control risk (fecteau et al., ) . the previous studies examined only immediate changes after training and the decision task selection typically included a single task. on the basis of previous studies, we chose the balloon analog risk task that has strong operability with initial results that are relatively stable and we appropriately increased the time interval between cognitive training and posttest decision-making task assessment. after excluding the immediate effects of training, we wanted to demonstrate that classic response inhibition training can also effectively change performance in this task. it can make the response suppression training more stable, more generalizable, and more convincing for improvements in risky decision-making. this paper proposes a hypothesis: classic response inhibition training can reduce risk behavior in the balloon simulation risk task, thereby resulting in more rewards. a total of university students were randomly divided into a training group ( men and women; mean age = . years, sd = . ) and a control group ( men and women; mean age = . years, sd = . ). there was no significant difference in age between the two groups [t ( ) = . , p = . ]. subjects were included based on the following inclusion criteria: they were - years old, physically and mentally healthy, right-handed, with normal vision or correctedto-normal vision, and had not participated in other relevant psychological experiments. the exclusion criteria were as follows: neurological disorders, alcohol or other substance abuse or overdependence, mental illness, and treatment with any psychotropic substance. the training process in our experiment is all person-by-person training. under the condition of limited manpower and financial resources, the workload of these subjects is close to the maximum. however, post hoc power calculation is calculated by gpower . . . with a sample size of participants, a significance level of % and an effect size of . . the calculated power value is . , proving that the sample size is sufficient. this research was approved and strictly implemented the recommendations of the local ethics committee. all subjects were given detailed experimental instructions and agreed to participate in the experiment. then, they signed the informed consent in accordance with the declaration of helsinki and were entitled to a certain payment after the experiment was completed. the training group adopted the go/nogo task and sst as training tasks that were presented using e-prime . . all experiment started with a short practice phase to make sure that the subjects understood the rules of the task completely. all training tasks were completed in the laboratory over a total of weeks. the two groups were assessed with the stroop task and the balloon analog risk task to evaluate the pretest and posttest performance on inhibitory control and risky decision-making tasks at weeks and . in the intervening weeks in the training group, two classic paradigms were simultaneously used and cross trained. the whole training schedule consisted of -min sessions, three times per week. during each training session, the two tasks alternated twice. in this study, two kinds of response inhibition training tasks were adopted. on the one hand, to increase the generalization effect of training, the inhibitory control ability was improved by automatic inhibition and top-down control inhibition (spierer et al., ) . on the other hand, with this kind of pure training, the more obvious the training boost will be and the more likely that the change in gambling task performance was due to improved inhibitory control. the control group read popular science articles related to self-control and were required to complete a task of summarizing the articles. the time and frequency for this task were consistent with the cognitive training of the training group. there were double triangles ("go") and single triangles ("nogo") used as stimuli in the task. the participants had to make a button-press response to the double triangle and inhibit their response to the single triangle. go and nogo stimuli were randomly presented in a : ratio. all stimuli were presented for ms in the center of the screen with a , ms interstimulus interval. the experimental phase consisted of go stimuli of trials and nogo stimuli of trials. there was a pause at the halfway point of this task and the participants could take a break or press any key to continue the experiment. the optimal performance of the task is to minimize the response time and the number of errors (the sum of the number of omission errors and commission errors). the task is the classic response inhibition task paradigm that has been widely used and is also recognized as a method that reflects inhibitory control abilities in a relatively simple and pure way (congdon et al., ; wostmann et al., ; enge et al., ) . the participants were required to press the "f " or "j" key when the "f " or "j" letter (go signal), respectively, appeared, and during a relatively low proportion of trials ( %) with obvious red dots (stop signal) appearing after the go signal, to immediately suppress the impulse to press the button. the task consisted of trials, including no-stop stimuli and stop stimuli. the center of the screen shows a fixation point (+) of ms before all the stimuli appear, and there is a , ms interval after the button response. in the no-stop stimulus, the most presentation time of the go signal was , ms. if the subject does not press the button in time, the screen will show "too slow". in the stop stimulus, the stop signal will appear later than the go signal and the stimulus presentation time is still , ms at most. if the subject does not immediately suppress the key, there will also be an interval of , ms. the difference between this task and the go/nogo task is that each stop signal is preceded by a reaction impulse and the clever experimental design allows a measure of behavioral inhibition time (verbruggen and logan, ; verbruggen et al., ; i.e., stop-signal reaction time, ssrt). the tracking method was used to automatically adjust the time when the stop signal appeared (i.e., stop-signal delay, ssd) and the initial value was set at ms. when the inhibition was successful, the ssd increased by ms, while the inhibition failed, and the ssd decreased by ms to ensure that the successful inhibition rate of the subjects was approximately equal to %. then, the ssrt value can be calculated (ssrt was equal to the average go reaction time minus the average ssd). the stroop task, which is commonly used for inhibitory control and relatively complicated in processing, was used to evaluate the ability of stimulus interference to inhibit or selectively focus on target-related stimuli (turner et al., ) . the participants were asked to select the corresponding key according to the four colors of red, blue, green, and yellow fonts. all stimuli were presented for ms followed by fixation point (+) for ms and there was a , ms interval after the subject responded. the task consisted of trials. four colors and four fonts were randomly matched and presented (every font was matched with four different colors, so that the ratio of consistent trials to inconsistent trials was : ) and consistent and inconsistent response times (rt) were recorded. the conflict effect (incongruent trials rt -congruent trials rt) and conflict score (conflict effect/congruent trials rt) were calculated to evaluate the two groups before and after the inhibitory control ability training (maraver et al., ) . the balloon analog risk task was used to evaluate risky behaviors and is a decision-making task that can effectively simulate realistic risky behaviors that are relatively stable under laboratory conditions (lejuez et al., ) . the experimental programming of this task was based on computer programming (c++) prepared and rendered on the computer screen. the participants can make money by inflating the balloon with a click of the mouse (earning yuan per inflation, which was included in the temporary account), but if the balloon bursts, they lose the money they made during the round (the temporary account). at the same time, the participants can choose to stop the pump at any time and the temporary account is transferred into the permanent account. each balloon is blown between and times and there is a predetermined explosion point (randomly set by the computer). the participants were asked to conduct balloon trials to make money and were given the sum from their permanent accounts for the balloon trials. the only way to make money is to stop the balloon before it explodes. the subjects were also told that the goal was to inflate the balloon as large as possible without exploding to maximize the benefit. the final benefit of each subject was recorded and average adjusted pumps (i.e., adj bart; adj bart = total number of unexploded balloon pumps/number of unexploded balloons) was calculated to measure the performance and impulsivity in the task. first, a curve was drawn between the performance in the two tasks in the training group and the training time. then, two independent sample t-tests were conducted on the pretest values of the stroop task and balloon analog risk task for the two groups and no significant difference was found between the two groups at pretest. because the experiment adopted a mixed design with between-and within-subjects factors, mixed-model anovas of (control group and training group) × (pretest and posttest) factors were used to evaluate the transfer effect of response inhibition training to stroop performance and its impact on balloon analog risk task performance. finally, we further analyzed the correlation between the initial threshold and the change amount of the training group. since both tasks were completed twice in one training session, we took the average of the two as the performance for that training session. repeated-measures anovas were conducted to compare the performance in the first session with that of the sixth session. as shown in figure , both go rt and ssrt were gradually reduced from the first to the last training session in their respective tasks and the differences reached statistical significance [go rt: f ( , ) = . , p < . , η = . ; ssrt: f ( , ) = . , p < . , η = . ]. in the two tasks, the error rate did not significantly change from the first to the sixth training session [go/nogo task: f ( , ) = . , p = . , η = . ; sst: f ( , ) = . , p = . , η = . ; figure ]. however, there was a downward trend in the go/nogo task, especially across the first four training sessions. the pretest conflict effect [t ( ) figure , we also found a significant time × group interaction effect [conflict effect: f ( , ) = . , p = . , η = . ; conflict score: f ( , ) = . , p = . , η = . ]. through simple effect analysis, two groups of effects were obtained. the conflict effect and conflict score in the training group after training were significantly lower (p < . ), while no significant difference between pretest and posttest performance was found in the control group [conflict effect: f ( , ) = . , p = . , η = . ; conflict score: f ( , ) = . , p = . , η = . ; table ]. the inhibitory control ability in the training group was improved compared with that of the control group because of the training. frontiers in psychology | www.frontiersin.org july | volume | article as shown in figure , there was no significant difference between the two groups in the pretest adj bart [t ( ) we further analyzed the correlation between the initial threshold and the change in the training group and found a significant negative correlation (reward: r = − . , p < . ; adj bart: r = − . , p < . ; figure ). the results indicated that adj bart and reward acquired by the training group after training significantly increased compared with that before training. moreover, the subjects with lower pretest indexes had a greater range of changes through training. from the results of the whole experiment, we not only improved the performance of the training task through weeks of response inhibition training (go/nogo and stop-signal tasks), but also more importantly, we found the migration effect of training in the untrained stroop task and balloon analog risk task. the fact that inhibition control plays an important role in people's complex decision-making process has been verified. there was a gradual improvement in the performance of the training group in two classic response inhibition tasks. the error rate in the go/nogo task (the ratio of the sum of omission errors and commission errors) showed a downward trend over the first four training sessions and increased in the later sessions. moreover, it can be seen from the results of the last two training sessions that the task response decreased while the error rate increased. this may have been because the participants were too reactive, which sometimes led to a rebound in error rates. in the later sessions, there was a gradual balance between the reaction time and the error number, and finally, it tended to be stable. therefore, although the error rate during the last four training sessions showed a slight upward trend, the overall response time showed a downward trend and an obvious training effect could still be seen. the error rate in the sst refers to the proportion of errors in the go response, not the proportion of suppression failures. the low error rate during the first exposure may have been due to the relatively small allocation of cognitive resources in the process of inhibition. with the increase in the allocation of cognitive resources in the inhibition process, the accuracy of keystrokes is ignored and the error rate changes little or slightly increases. however, we can still see the improvement in task performance from the trend in the ssrt scores. there was a significant difference in the effect of the two kinds of training, and it was also found that the pursuit of reaction speed might lead to a decrease in accuracy in the later periods of training, and eventually, the two tended to stabilize. in addition, enge et al. also found that in the latter stage of training in the sst, ssd and mean go reaction time were simultaneously reduced, which would eventually lead to the reverse increase in ssrt (enge et al., ) . in this experiment, this phenomenon occurred in some subjects, but the overall trend was not found. lower conflict scores in the stroop task in the training group after training suggested that response inhibition training could be transferred to interference control. this also showed that the two classic response inhibition task training methods are effective. friedman and miyake used latent variable analysis to demonstrate that almost all inhibition tasks have a common inhibitory control mechanism (friedman and miyake, ) . although brydges et al. ( ) later used erp technology to show that the two tasks engaged two different cognitive components (brydges et al., ) , they were still closely related, and performance could be transferred (maraver et al., ) . from the adj bart in the balloon analog risk task, it can be concluded that the training of response inhibition led to an increase in the subjects' risky behaviors, which seems to contradict our hypothesis. however, at the same time, the reward acquired in the task increased after the training. according to the value gained by pumping up the balloon and the probability of explosion, the value of the first rounds of pumping is greater than the value of the nonpumping rounds. it is not until the seventeenth turn that pumping up the balloon becomes irrational (lejuez et al., ) . therefore, from the perspective of profits obtained, the subjects were too conservative to avoid balloon explosion, thereby losing the chance to win more money before training. this also explained our increased risk-taking behavior and benefits after training. we therefore suggest that the key decision for balloon analog risk task is not to inflate (this is a continuous process) but to decide when to stop inflating and put the contents of the temporary account into the permanent account. only by making a rational decision to stop inflating (properly suppressing decisions that are too early or too late) can the maximal amount of reward be obtained. each inflation is actually equivalent to a go reaction, and it is the ability of response inhibition that is needed as the basis for the critical and appropriate stopping of inflation. the results of increasing risk-taking behaviors in this paper were inconsistent with those of the previous study (verbruggen et al., ) . although many gambling tasks are task paradigms for evaluating risky decision-making, different tasks represent different risky decision-making processes (buelow and blaine, ) . the stopping of inflation in the balloon analog risk task may be more related to the ability to response inhibition, which may also have contributed to the inconsistent results across different tasks. we further analyzed the significant negative correlation between the initial threshold and the change amount in the training group and found that the lower the initial value was, the more significant the improvement. this suggested, to some extent, that people with lower initial values were more likely to improve (schmaal et al., ) . therefore, we should probably focus more on the lower level of the population, where the limited training intensity could achieve a higher training effect. there are many other factors that affect inhibitory control training. it is widely recognized that emotion and motivation affect cognitive inhibition processes, higher decision-making processes, and other neural or psychological functions (padmala and pessoa, ; turner et al., ) . in the traditional sense, it is considered that subcortical structures, such as the amygdala, ventral striatum, and hypothalamus, are mainly responsible for processing emotions and behaviors, while cortical structures, such as the dorsolateral prefrontal cortex and anterior cingulate cortex, are responsible for activating cognitive control and higher executive functions (pessoa, ) . therefore, appropriate incentives and positive feedback will effectively improve the training effect of the subjects. in addition, some studies failed to achieve transfer effects of training (enge et al., ; zhao et al., ; kable et al., ) , which may be because the differences were not significant due to the insufficient number of subjects or the training time and intensity did not reach the threshold needed to transfer tasks (kable et al., ) . as shown in figure , there were also some subjects with opposite results, but this pattern of results were mainly concentrated in those with higher starting values. if the primary group chosen were primarily high-level people, then grouplevel indifference would be inevitable. therefore, the initial level of grouping will also affect the training effect. the basis for improving decision-making through training in response inhibition is brain plasticity (i.e., a change in behavior and its underlying brain anatomy based on experience; spierer et al., ) . these changes can facilitate the acquisition of new skills, the improvement of acquired abilities, and the recovery of defective or impaired functions (kelly and garavan, ) . changes in behavior and brain plasticity induced by training have been demonstrated at different levels of executive function. research has shown that people's inhibitory control ability is closely related to the inferior frontal gyrus and dorsolateral prefrontal cortex (aron et al., ) . at the same time, these brain regions also play an important role in risky decision-making tasks (chiu et al., ) . during the training of the inhibitory control tasks, the corresponding brain regions will be repeatedly activated and the connections between the corresponding brain regions will be increased, which will inevitably affect the neural connections in the decisionmaking process. although there is no neuroscientific evidence, it is likely that this is one of the important reasons that response inhibition training changes subsequent performance in decision-making tasks. the results of the control group also showed that reading about self-control skills alone was not enough to improve the participants' inhibitory control skills, which also reflected the need for cognitive training of response inhibition. first, the selection range of the subjects was relatively limited, leading to limited generalization. second, the training time was short, and there was no long-term tracking due to the effect of novel coronavirus, so the duration of the transfer effect cannot be determined at present. third, the degree of improvement in the balloon analog risk task performance in the training group was relatively limited, which may also be related to shorter training time and lower intensity. fourth, as the training group needs to spend a lot of time in the whole training process, our subjects may be potentially inadequate. the problem of subject size is also a shortcoming of most cognitive training studies. because of this, slight differences in some experimental conditions may lead to different migration outcomes or no migration effects between many similar training studies. in addition, for the measurement of inhibitory control and risky decision-making, a variety of evaluation indicators should be used, such as questionnaires, behavioral observation, and imaging techniques such as erps and magnetic resonance imaging (mri), rather than cognitive task paradigms on computers. a single score from the inhibition task or risky decision-making task cannot represent the complex control processes that may be correlated with each other, so it is necessary to use multiple tasks to evaluate the inhibitory control ability and risky decision-making (dougherty et al., ) . this study confirms that classic response inhibition training can increase risk-taking behavior in the balloon analog risk task, improve their overly conservative behaviors, properly inhibit them to obtain more benefits, and substantially increase economic rationality. during the whole experiment, various experimental conditions were strictly controlled and the training and transfer effects were statistically significant. it is particularly important that compared with inhibiting the near transfer between control tasks, the risky decision-making task can be considered a far transfer (crespi et al., ) and this experiment is a good attempt at selecting a cognitive training far transfer task. research on the transfer effect of inhibitory control training to higher cognitive function and the tracking of training will also become the focus of future research in this field. however, at the same time, we must also make it clear that these higher cognitive processes are not just inhibitory control processes, and whether there is a more general, fundamental process is debatable. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by air force medical university. the patients/ participants provided their written informed consent to participate in this study. px completed the experiment and wrote it. dw and yc assisted the experiment and analyzed the data. zw provided technical guidance and site support. wx grasped the idea and financial support. all authors contributed to the article and approved the submitted version. inhibition and the right inferior frontal cortex behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of adhd a systematic review of the relationship between eating, weight and inhibitory 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differences reliability and plasticity of response inhibition and interference control wesley says": a children's response inhibition playground training game yields preliminary evidence of transfer effects key: cord- -s j naz authors: sundaram, maria e.; mcclure, david l.; vanwormer, jeffrey j.; friedrich, thomas c.; meece, jennifer k.; belongia, edward a. title: influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness date: - - journal: clin infect dis doi: . /cid/cit sha: doc_id: cord_uid: s j naz background. the test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve). recent studies have suggested that influenza vaccination increases the risk of noninfluenza respiratory virus infection. such an effect could create bias in ve studies using influenza-negative controls. we investigated the association between influenza infection, vaccination, and detection of other respiratory viruses among children < years old and adults ≥ years old with acute respiratory illness who participated in seasonal studies of influenza vaccine effectiveness. methods. nasal/nasopharyngeal samples collected from – through – were tested for respiratory virus targets using a multiplex reverse-transcription polymerase chain reaction (rt-pcr) platform. vaccination status was determined using a validated registry. adjusted odds ratios for influenza and vaccination status were calculated using three different control groups: influenza-negative, other respiratory virus positive, and pan-negative. results. influenza was detected in % of children and % of adults. noninfluenza respiratory viruses were detected in % of children and % of adults without influenza. the proportion vaccinated did not vary between virus-positive controls and pan-negative controls in children (p = . ) or adults (p = . ). influenza infection was associated with reduced odds of vaccination, but adjusted odds ratios differed by no more than . when the analysis used influenza-negative or virus-positive controls. conclusions. influenza vaccination was not associated with detection of noninfluenza respiratory viruses. use of influenza-negative controls did not generate a biased estimate of vaccine effectiveness due to an effect of vaccination on other respiratory virus infections. the case vs test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve) [ ] [ ] [ ] [ ] [ ] [ ] . cases and controls are recruited at the time of presentation of acute respiratory illness (ari) in clinic and hospital settings. individuals presenting with ari who test positive for influenza are considered cases, whereas those who test negative for influenza are considered controls. this study design is convenient to implement and inherently accounts for potential confounding due to differences in healthcareseeking behavior between vaccinated and unvaccinated individuals [ ] [ ] [ ] . it has recently been suggested that influenza vaccination may increase the risk of non-influenza respiratory virus infection by decreasing temporary nonspecific immunity [ , ] . one proposed mechanism involves activation of the innate immune response following influenza infection, leading to a temporary reduction in the risk of infection with a different respiratory virus. by reducing the risk of influenza infection, the influenza vaccine could paradoxically create an increased risk of infection with other noninfluenza respiratory viruses. if this phenomenon occurs, it could lead to biased estimates of influenza vaccine effectiveness in studies using laboratory-confirmed influenza cases and influenza-negative controls. in this scenario, the risk of noninfluenza viral illness would be higher in vaccinated than unvaccinated individuals, and an 'influenza-negative' control group would therefore have a higher proportion of vaccinated individuals compared to the source population. this could theoretically contribute to overestimation of true ve (ie, bias away from the null); therefore, a key assumption of the test-negative control design of influenza vaccine effectiveness studies is that the proportion of noninfluenza viral illness does not differ by influenza vaccination status [ ] . the goals of this study were to determine if influenza vaccination is associated with detection of noninfluenza respiratory viruses and to determine if vaccine effectiveness estimates differ when different control groups are used in the analysis. to achieve these goals, we analyzed available data from members of a community cohort who saw a physician for acute respiratory illness and consented to participate in a study of influenza vaccine effectiveness over influenza seasons. the vaccine effectiveness study enrolled individuals of all ages (with some variation by season), but this analysis was restricted to children < years old and adults ≥ years old. for participants in these age groups, multiplex reverse transcription polymerase chain reaction (rt-pcr) testing was subsequently performed to detect other respiratory viruses, providing an opportunity to investigate the relationship between influenza vaccination and infection with other viral pathogens. young children and older adults are among the most vulnerable individuals to influenza infection and complications [ , ] and calculating influenza vaccine effectiveness in these groups is therefore of high importance [ ] . the marshfield clinic research foundation has conducted seasonal studies of influenza vaccine effectiveness in a wisconsin population cohort since the - season. the details of the seasonal studies have been reported elsewhere [ , ] . briefly, patients with ari were recruited during each influenza season in primary care clinics, urgent care, emergency department, and an acute care hospital. symptom eligibility criteria varied by season but included fever/feverishness or cough during most seasons. individuals with illness duration ≥ days ( - through - ) or > days ( - through - ) were excluded to minimize false negative rt-pcr results. after obtaining informed consent, a nasal swab (children < years old) or a nasopharyngeal swab (adolescents and adults) was obtained and placed in viral transport media for influenza testing. symptoms and onset date were assessed during the enrollment interview. real-time rt-pcr was performed each season to identify influenza cases. after testing was complete, aliquots of samples in viral transport media were frozen at − °c. this study was reviewed and approved by the marshfield clinic institutional review board. during each season, all study participants (or parents) provided informed consent for influenza testing. multiplex rt-pcr testing to detect additional viruses was subsequently approved by the irb with a waiver of informed consent. archived samples were tested for the presence of respiratory virus nucleic acid using a multiplex respiratory virus panel (genmark dx esensor respiratory viral panel). this multiplex panel tested for respiratory syncytial virus (rsv) a and b, human rhinovirus, human metapneumovirus, parainfluenza viruses - , influenza a and b (including subtypes of influenza a), coronaviruses oc , nl , hku , and e, and adenoviruses b and e. nucleic acid was extracted from the swabs using the roche magnapure . system and was then amplified using rt-pcr with target-specific primers. target-specific signals were determined by voltammetry, a process that generates electrical signals from ferrocene-labeled signal probes. the multiplex assay has been validated for influenza and rsv a and b against singleplex assays approved by the centers for disease control and prevention (cdc) and found to have %, %, and % sensitivity and %, %, and % specificity for influenza a, influenza b, and rsv, respectively (unpublished data). cases were defined as laboratory-confirmed influenza based on a positive real-time rt-pcr test for influenza a (h n ), a (h n ), a (h n )pdm , or type b. three different control groups were constructed for analytical purposes. control group included all individuals testing negative for influenza (influenza-negative controls); group included individuals negative for influenza but positive for at least one other respiratory virus in the multiplex panel (virus-positive controls); and group included individuals negative for all respiratory viruses in the multiplex panel ( pan-negative controls). the first control group is the standard definition used in test-negative case-control designs, whereas the second and third control groups represent subsets of the first group. vaccination status and dates were determined by a real-time, internet-based registry used by all immunization providers serving the local population (www.recin.org). cases and controls were considered vaccinated if seasonal trivalent inactivated influenza vaccine (tiv) or live attenuated influenza vaccine (laiv) was received at least days before ari symptom onset. children who were partially vaccinated according to the advisory committee on immunization practices (acip) definition (ie, received of recommended doses) were excluded [ ] . separate analyses were conducted for children < years old and adults ≥ years old, and only the first enrollment was included for each individual in a given season. vaccination status was compared for virus-positive controls and pan-negative controls using logistic regression. odds ratios describing the association between vaccination and influenza infection were determined separately for each of the case-control groups by logistic regression. logistic regression models included influenza status as the outcome and vaccination status as the primary exposure variable with adjustment for potential confounders, including gender, influenza season, age (continuous), presence of a chronic disease conferring increased risk for influenza complications [ ] , and interval in days from symptom onset to swab collection. sas . (sas institute, cary, nc) was used for all statistical analyses. over influenza seasons, children and adults contributed and unique observations, respectively. influenza was detected in ( . %) children and ( . %) adults. among those without influenza, a respiratory virus was detected in ( . %) of children and ( . %) of adults. in children without influenza, the most commonly detected single viruses were rsv (n = ), human rhinovirus (n = ), human metapneumovirus (n = ), and parainfluenza (n = ); ( . %) of swabs from children were positive for ≥ noninfluenza respiratory viruses. in children, the most common coinfection was rsv with rhinovirus (n = ). in adults without influenza, the most commonly detected single viruses were rsv (n = ), human rhinovirus (n = ), human metapneumovirus (n = ), and coronavirus (n = ); ( . %) of swabs from adults were positive for ≥ noninfluenza respiratory viruses. in adults, the most common coinfection was rsv with coronavirus (n = ). the virus-positive controlgroup and pan-negativecontrolgroup did not differ with regard to age, gender, vaccination status, or interval from symptom onset to swab in either children or adults ( table ). the proportion of children with a high-risk health condition did not differ between control groups; there were more adults with a high-risk health condition in the pan-negative control group than in the virus-positive control group (table ) . in univariate analyses, no association was found between influenza vaccination and single virus detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. single infection with parainfluenza virus was less common in vaccinated children ( . %) than vaccinated children ( . %; p = . ). among adults, there was a significant association in the opposite direction: parainfluenza was detected in . % of vaccinated and . % of unvaccinated adults (p = . ). for both children and adults, the adjusted odds of influenza infection were significantly lower in the vaccinated group relative to any of the control groups, indicating significant vaccine effectiveness ( table ). among adults, the adjusted odds ratio (aor) ranged from . to . ; among children the aor ranged from . to . depending on which control group was used. in both children and adults, the aor using 'influenza-negative controls' (ie, the current standard for ve studies) varied by no more than . compared to the aor using other virus-positive controls, and all aor confidence intervals overlapped. the adjusted odds of being vaccinated did not differ significantly in the pan-negative control group vs the other virus-positive control group, either in children (p = . ) or in adults (p = . ). results were similar when the analysis was repeated after excluding children who received laiv. there were no adults in this analysis who received laiv. detection of a noninfluenza respiratory virus by multiplex rt-pcr was not associated with influenza vaccination status over a period of six influenza seasons. in both children and adults, the virus-positive control group and the pan-negative control group were similar in terms of age distribution, gender, and influenza vaccination status. there was no association between influenza vaccination and detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. only parainfluenza infection was significantly associated with influenza vaccination, but the association was in opposite directions for children and adults. the p values were not adjusted for multiple comparisons, and the inconsistency between children and adults suggest that this association is the result of a type error. to assess the potential impact of noninfluenza respiratory viruses in vaccine effectiveness studies, we calculated the aor for influenza vaccination in cases and controls using different control groups. among children, the aors were . and . , respectively, using influenza-negative controls and virus-positive controls. this corresponds to an absolute difference of % in the vaccine effectiveness estimate, with broadly overlapping confidence intervals. nearly all vaccinated children in this analysis received tiv, and the results were similar when laiv recipients were excluded. among adults ≥ years old, the adjusted odds ratios were nearly identical ( . and . , respectively) when using influenza-negative controls and viruspositive controls, with overlapping confidence intervals. other studies have found evidence of an association between influenza vaccination and infection with a non-influenza respiratory virus. in , a study of influenza vaccine effectiveness in australian children to months old found that influenza vaccination was associated with detection of other respiratory viruses [ ] . nasal swabs from children were tested by rt-pcr for influenza, rhinoviruses, rsv, parainfluenza virus - , human metapneumovirus, and enteroviruses. adjusted vaccine effectiveness was % ( % ci, - ) using influenza-negative controls and % ( % ci, - ) using other virus-positive controls. the authors noted the higher point estimate of vaccine effectiveness using virus-positive controls, although there was substantial overlap in the confidence intervals for these point estimates. the authors speculated that the difference between point estimates could be due to false negative rt-pcr tests in some children (ie, pan-negative controls) due to inadequate sample collection. they also mentioned the possibility that influenza vaccination could increase the risk of infection with other viruses, but they viewed this as biologically implausible. the relationship between influenza vaccination and infection with noninfluenza viruses was also investigated in a clinical trial of tiv in children [ ] . in that study, tiv recipients (n = ) had a -fold increase in risk of infection with noninfluenza respiratory viruses, compared to placebo recipients (n = ); the incidence of seasonal influenza did not differ significantly between the tiv and placebo groups. the authors acknowledged that the association with noninfluenza viruses could be spurious, but they suggested that 'receipt of tiv could increase influenza immunity at the expense of reduced immunity to noninfluenza respiratory viruses, by some unknown biological mechanism' [ ] . they identified temporary nonspecific immunity as a possible explanation for this observation. according to this proposed mechanism, unvaccinated children were more likely to acquire influenza infection, and the abbreviations: aor, adjusted odds ratio; ci, confidence interval; uor, unadjusted odds ratio. a all models adjusted for gender, influenza season, age (in years), presence of high-risk health condition, and interval (days) from symptom onset to swab collection. resulting innate immune response provided temporary, nonspecific protection against infection with other respiratory viruses. this was described further in a letter that raised concerns about bias in studies of vaccine effectiveness that use rt-pcr confirmed cases and influenza-negative controls [ ] . these studies had relatively small sample sizes and were limited to a single influenza season. however, one of the studies was a randomized, placebo-controlled trial, a study design that is less susceptible to bias and confounding compared to observational studies. we were unable to replicate the previously reported association in children or adults with a larger sample size over multiple influenza seasons. however, we cannot rule out the possibility that vaccination may alter susceptibility to non-influenza viruses in some circumstances. there is limited biological evidence to support an effect of nonspecific immunity across virus families or species and limited evidence to support the role of vaccination in such immunity. cross-reactivity of the t-cell response has been described before for different subtypes of influenza a [ ] [ ] [ ] , although cross-reactivity generated by the innate immune system likely lasts only - days [ ] and may not apply to interactions between viruses of different families or species. other researchers have also suggested that outbreaks of different respiratory viruses may interact or interfere with each other on a population level [ ] [ ] [ ] . this mechanism may help to explain effects not seen in our analysis but which have been reported elsewhere [ , ] . in conclusion, our findings do not support the hypothesis that influenza vaccination is associated with an increased risk of infection with noninfluenza respiratory virus. further research may aid in determining the biological plausibility of temporary nonspecific immunity generated by infection with specific respiratory viruses. finally, we found no difference in vaccine effectiveness estimates using test-negative vs other virus-positive control groups. the results of this analysis strongly support the validity of case vs test-negative control study designs that 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t-cell immunity to influenza immunity to respiratory viruses interference between outbreaks of respiratory syncytial virus and influenza infection interference between outbreaks of epidemic viruses does viral interference affect spread of influenza? acknowledgments. we gratefully acknowledge the staff of data managers, research coordinators, interviewers, laboratory technicians, and others who contributed to the vaccine effectiveness study and this analysis. we also thank alicia fry, mark thompson, and frank sifakis for manuscript review and feedback.financial support. medimmune, llc, provided funding for multiplex reverse-transcription polymerase chain reaction testing.potential conflicts of interest. m. e. s., j. k. m., and e. a. b. receive research funding from medimmune, llc. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -dkbujz authors: chughtai, abrar a.; seale, holly; islam, md saiful; owais, mohammad; macintyre, c. raina title: policies on the use of respiratory protection for hospital health workers to protect from coronavirus disease (covid- ) date: - - journal: int j nurs stud doi: . /j.ijnurstu. . sha: doc_id: cord_uid: dkbujz nan novel coronavirus (covid- ) emerged in china in december and as of rd march , more than , cases and deaths have been reported from countries ( world health organisation (who) ). most of the cases and deaths have occurred in wuhan city in china where the outbreak started. as drugs or vaccines are not yet available, various non-pharmaceutical measures have been recommended to reduce the spread of infection, including hygiene and disinfection, improving environmental control, early detection and reporting, isolation, quarantine, use of personal protective equipment, social distancing and travel restrictions (( world health organization (who) ; bell et al., ) ). in most pandemic plans, tiered strategies which target front line health workers first, followed by the general community, are usually defined. yet many of these strategies have some level of controversy attached and lack a large evidence base . some are starting to be phased out in some settings such as the use of thermal scanners at airports, due to a lack of evidence, especially for infections with pre-symptomatic transmission ( gunaratnam et al., ) . however, the use of other interventions continues to be debated. the lack of agreement on the selection and use of masks (medical or surgical facemasks) and respirators (n /p /ffp or equivalent) is reflected in inconsistent and conflicting policies worldwide chughtai et al., ) . masks and respirators are commonly used to protect healthcare workers from respiratory infections, particularly during the initial periods of outbreaks/ pandemics when other control measures not yet available ( aiello et al., a; aledort et al., ) . in this paper we examined existing policies of selected health organization and countries on the use of masks and respirators to protect hospital health workers from covid- . while there is a high level of agreement amongst key agencies that masks and respirators play a role in the protection of health workers, there are currently discrepancies between these agencies regarding how and when the different products are used. the world health organization (who), the us centers for disease control and prevention and other leading health organisations have different recommendations for the selection of respiratory protec-tion. for example, the who recommends using masks to protect health workers from covid- during routine care and respirators during aerosol generating procedures ( world health organisation (who) ). in contrast, the us centers for disease control and prevention ( center for disease control and prevention (cdc) ) and the european center for disease prevention and control ( european centre for disease prevention and control (ecdc) ) recommend using respirators during both routine care of covid patients and high risk situations. individual countries also have different policies; some are in line with the who, and others with the us centers for disease control and prevention. for example, the australian ( communicable diseases network australia (cdna) ) and canadian guidelines ( government of canada ) align with the who, while uk ( public health england ) and chinese guidelines ( chinese center for disease control and prevention ) align with the us centers for disease control and prevention and european center for disease prevention and control. while all organisations recommend using n / p /ffp or equivalent respirators, public health england (uk) recommends using filtering facepiece (ffp ) respirators for all cases and the european center for disease prevention and control recommends using ffp during aerosol generating procedures. australian guidelines also recommend powered air purifying respirators while performing aerosol generating procedures on multiple patients. although us centers for disease control and prevention guidelines also discuss powered air purifying respirators indication of their use is not clear. these conflicting policies may cause confusion for hospital managers, nurses and other health workers. conflicting guidelines have resulted in a large amount of online debate between infectious disease providers and infection prevention control experts about what approaches should be adopted. history is repeating itself with the same controversies around powered air purifying respirators during the the only similarity is that all health organisations and countries generally recommend the use of masks by suspected or infected cases of covid- (i.e. source control) to prevent spread of infections. they simultaneously state that community use of masks has no benefit. yet there are more randomised controlled clinical https://doi.org/ . /j.ijnurstu. . - /© elsevier ltd. all rights reserved. trials (rcts) supporting the use of masks in the community than for source control. the community trials show a benefit of face masks with or without hand hygiene, conditional on compliance ( simmerman et al., ; aiello et al., ; aiello et al., b; suess et al., ; macintyre et al., ; cowling et al., ). there are only two rcts of clinical efficacy of source control ( canini et al., ; macintyre et al., ) , and one small experimental rct of subjects ( johnson et al., ). these suggest a benefit, but larger trials are needed. the main difference in masks and respirators is their intended use. masks were originally designed to prevent spread of infections from wearers to other people around them, referred to as "source control". they are also used to protect from infections transmitted through the droplet mode and splashes or sprays of blood or body fluids. disposable medical or surgical masks are common types of face masks used by both health workers and general public. in contrast to this, respirators are designed for respiratory protection. a medical or surgical mask may be enough to prevent droplet transfer, while a respirator is required for airborne infection. in terms of mask use, the physical barrier may also prevent contact transmission such as hand to face/mouth/nose. a respirator may provide protection against multiple modes of transmission, including droplet, airborne and hand-to-mouth/nose transmission. whilst the relative contribution of each mode is difficult to quantify, clinically, the debate about the modes of transmission is academic if an intervention is shown to prevent infection. like other coronavirus diseases (e.g. sars and mers), covid- is believed to be transmitted through droplet and contact modes however other transmission modes, such as airborne, are likely given the virus is found in higher concentrations in the lungs than the upper respiratory tract ( world health organisation (who) ). there had been evidence of airborne transmission of sars as well, therefore respirators were recommended for sars during - outbreak ( mckinney et al., ) . in canada, initially masks were recommended, but this recommendation was later changed to respirators due to the deaths of health workers. a recent study demonstrated the presence of coronavirus in anal swabs from infected patients and possibility of transmission through faecal-oral route ( zhang et al., ) . transmission dynamics for covid- are still unclear and pharmaceutical control measures are not yet available, therefore n or higher respirators should be offered to health workers who are working at the frontline ( macintyre et al., a; macintyre et al., b). health workers and other first responders in high coronavirus transmission areas (e.g. wuhan) should use respirators during routine care of coronavirus cases. health workers and first responders in low risk countries should use a respirator when encountering a suspected or confirmed case of coronavirus. if respirators are not available, then masks should be used. extended use and reuse of masks and respirators was a common practice during past epidemics and pandemics due to shortage of products ( chughtai et al., ; beckman et al., ; lautenbach et al., ; rebmann and wagner, ). according to the us centers for disease control and prevention, extended use refers to "the practice of wearing the same n respirator for repeated close contact encounters with several patients, without removing the respirator between patient encounters" ( center for disease control and prevention (cdc) a). the us centers for disease control and prevention defines re-use as "the practice of using the same n respirator for multiple encounters with patients but removing it ('doffing') after each encounter" ( center for disease control and prevention (cdc) a). shortages of respirators were reported in many hospitals in us and japan during the influenza h n pandemic and staff had to use medical masks ( lautenbach et al., ; rebmann and wagner, ; tomizuka et al., ) . currently, the single use of medical mask and ffp respirators is recommended, but this is not always feasible. during a pandemic or extended outbreak, medical masks and ffp respirators may not be available for everyone. therefore the us centers for disease control and prevention and other health organisations have previously considered the extended use and re-use of medical masks and respirators during outbreaks, pandemics and other high demand situations (( center for disease control and prevention (cdc) a; occupational safety and health administration (osha) ; institute of medicine (iom) national academy of sciences )). during recent covid- epidemics the shortage of masks and respirators have been reported from many countries. however, the outer surface of medical masks or respirators may be contaminated and may be a source of infection (( institute of medicine (iom) national academy of sciences ; viscusi et al., ) ). a recent study showed that pathogens may be present on the outer surface of around % masks and risks increase with prolonged mask use ( chughtai et al., ) . the number of viral particles and length of survival are important factors to consider in case re-use is deemed essential ( institute of medicine (iom) national academy of sciences ). currently there is a lack of data regarding the period for which the same mask or respirators may be continuously used, and none of guideline address this. available data suggest that respirators may be used intermittently or continuously for around eight hours ( center for disease control and prevention (cdc) b) and that adverse effects of facemasks increase with more than eight hours use ( shenal et al., ) . it has been suggested that extended use of facemasks is acceptable if the mask is not wet and soiled. however there are currently no clinical studies supporting this practice (( occupational safety and health administration (osha) ; sonoma county department of health services )). policies and guidelines should also mention implementation of a comprehensive respiratory protection program for respirator use, which includes selection of certified respirators, training and fit checking and testing, and inspection, maintenance and storage ( occupational safety and health administration (osha) ). certified respirators should be used in healthcare settings and certification processes should be managed by a regulatory body, for example in the us the national institute for occupational safety and health regulates the certification process under regulation cfr ( national institute for occupational safety and health (niosh) ). similarly, in europe, the european norm standard ( european directive ) and in australia, as/ nzs standard regulates respirator use ( standards australia limited/standards new zealand ). all guidelines, except the who and the australian guidelines, briefly discuss the need for fit testing but do not provide detail on fit testing procedures. the who and the australian guidelines do not mention fit testing and instead mention fit check (or seal-check) which is not equivalent to fit testing. all guidelines however highlight the importance of training for respiratory (and other personal protective equipment) use. summing up, in the case of serious emerging infections, like covid- , the precautionary principle should be used for frontline health workers and a properly fitted respirator should be used. if respirators are not available, masks should be used. extended use and reuse are high risk practices and may lead to selfcontamination to the wearer and should be avoided. in case of shortage, extended use should be balanced against the risk of infections and the wearer should not remove masks between patients encounters. there should be a uniform policy around the use of personal protective equipment to avoid confusion which places occupational health and safety of health workers as a high priority. abrar ahmad chughtai had testing of filtration of masks by m for his phd more than years ago. m products were not used in his research. he also has worked with cleanspace technology on research on fit testing of respirators (no funding was involved). c raina macintyre receives funding from nhmrc (center for research excellence and principal research fellowship) and sanofi currently. she has received funding from m more than years ago for face mask research. there was no funding involved. research findings from nonpharmaceutical intervention studies for pandemic influenza and current gaps in the research facemasks, hand hygiene, and influenza among young adults: a randomized intervention trial mask use, hand hygiene, and seasonal influenza-like illness among young adults: a randomized intervention trial non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base evaluation 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examine medical mask use as source control for people with respiratory illness - quiz - . national institute for occupational safety and health (niosh), . niosh guide to the selection and use of particulate respirators . occupational safety and health administration (osha). u.s. department of labor. pandemic influenza preparedness and response guidance for healthcareworkers and healthcare employers covid- : infection prevention and control guidance infection preventionists' experience during the first months of the novel h n influenza a pandemic discomfort and exertion associated with prolonged wear of respiratory protection in a health care setting findings from a household randomized controlled trial of hand washing and face masks to reduce influenza transmission in pandemic influenza -phase infection control recommendations template standards australia limited/standards new zealand. respiratory protective devices. australian/new zealand standard. as/nzs the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial insufficient preparedness of primary care practices for pandemic influenza and the effect of a preparedness plan in japan: a prefecture-wide cross-sectional study evaluation of five decontamination methods for filtering facepiece respirators . novel coronavirus ( -ncov) situation reports available from infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care . world health organisation (who) . novel coronavirus ( -ncov molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes md saiful islam, mohammad owais school of public health and community medicine key: cord- -aq b authors: pettus, katherine; cleary, james f.; de lima, liliana; ahmed, ebtesam; radbruch, lukas title: availability of internationally controlled essential medicines in the covid- pandemic date: - - journal: j pain symptom manage doi: . /j.jpainsymman. . . sha: doc_id: cord_uid: aq b section of the world health organization (who) model list of essential medicines includes opioid analgesics formulations commonly used for the control of pain and respiratory distress, as well as sedative and anxiolytic substances such as midazolam and diazepam. these medicines, essential to palliative care, are regulated under the international drug control conventions overseen by united nations (un) specialized agencies and treaty bodies and under national drug control laws. those national laws and regulations directly affect bedside availability of internationally controlled essential medicines (icems). the complex interaction between national regulatory systems and global supply chains (now impacted by covid- pandemic) directly affects bedside availability of icems and patient care. despite decades of global civil society advocacy in the un system, icems have remained chronically unavailable, inaccessible and unaffordable in lower-and-middle income countries, and there are recent reports of shortages in high income countries as well. the most prevalent symptoms in covid- are breathlessness, cough, drowsiness, anxiety, agitation and delirium. frequently used medicines include opioids such as morphine or fentanyl and midazolam, all of them listed as icems. this paper describes the issues related to the lack of availability and limited access to icems during the covid- pandemic in both intensive and palliative care patients in countries of all income levels and makes recommendations for improving access. section two of the world health organization (who) model list of essential medicines includes opioid analgesics formulations commonly used for the control of pain and respiratory distress, as well as sedative and anxiolytic substances such as midazolam and diazepam ( ) . as these essential palliative care medicines are regulated under the international drug control conventions overseen by united nations (un) specialized agencies and treaty bodies ( ) and under national drug control laws, we refer to them as internationally controlled essential medicines (icems). the complex interaction between regulatory systems and global supply chains, as well as limited training, affect availability of icems and patient care. the lancet commission on pain and palliative care reported that of the total number of tons of morphine-equivalent opioids distributed in the world per year, only . metric tons is distributed to low-income countries (lics), reflecting a huge abyss is access to relief and resulting in massive, avoidable suffering ( ). while several authors, multilateral agencies and academia have used other different methods to estimate the actual need vs the reported consumption, these all demonstrate similar inequities in availability and access ( , ) . all these methods have been used for statistical purposes only and not as measures of therapeutic appropriateness. despite decades of global civil society advocacy in the un system, icems have remained chronically unavailable, inaccessible and unaffordable in low-and-middle income countries (lmics). this in spite of the fact that the who and other un treaty bodies recognize palliative care and pain relief as elements of the right to health ( ) . shortages in high income countries have recently been reported as well, due to the increase in demand as a result of the covid- pandemic ( ) . the authors, along with representative of other palliative care organizations and academia, have long advocated for the principle of "balance," which represents the dual obligation of governments to establish a system of control that ensures the adequate availability of controlled substances for medical and scientific purposes, while simultaneously preventing their non-medical use, diversion and trafficking, two primary goals of the international control system. ( ) pettus, k this paper describes the issues related to the lack of availability and limited access to icems in during the covid- pandemic in both intensive and palliative care patients in countries of all income levels and makes recommendations for improving access. palliative care providers in lmics facing the pandemic with already low stocks and fragile importation, production and supply chains, as well as unduly restrictive regulations controlling icems. for several years, even before the covid- pandemic, the incb has reported that the availability of these medicines is low to inadequate, ( ) and in response to the pandemic, recently published a press release calling on governments to take the necessary steps to ensure continued access to controlled medicines for pain relief and palliative care and for mental health and neurological conditions by fast tracking shipments of controlled medicines to health systems and supporting appropriate training of healthcare providers ( ). events, which provides strategic leadership for urgent and coordinated action on shortages within the eu in this pandemic, has set up with the pharmaceutical industry a system to fasttrack interaction on shortages between industry and the eu executive steering group ( ) . the most prevalent symptoms in covid- are breathlessness, cough, drowsiness, anxiety, reporting that the increase in the number of patients requiring ventilation has resulted in an increase in the demand for several opioids, some of which were already in shortage prior to the covid- outbreak. ( ) they request that supply be rapidly increased to ensure that hospitals can access the medications they need to treat covid- patients. in addition, public health experts and physicians recently asked the governors of us states that still practice the death penalty to release their stockpiled drugs in order to prioritize the needs and lives of patients ( ) . and in the uk, the national health service is reportedly working to increase stocks while at the same time facing many challenges. ( ) about countries with low and inadequate access to icems, we urge representatives of palliative care associations in all countries currently coping with, or preparing for this or for future pandemics, to contact their health ministries and national competent authorities to ensure that they are aware of the incb recommendations mentioned in its press release cited above. these include ensuring the maintenance of sufficient buffer stocks and using simplified control procedures for the export, transportation and provision of icems. . we urge palliative care associations to consider recommending their national competent authorities to utilize pooled procurement mechanisms such as the pan american health organization strategic fund, the pharmaceutical procurement service in the organization of eastern caribbean states, the gulf corporation council, and unicef and to resupply and build buffer stocks, as the markets will take advantage of increased demand to raise prices. the human rights council, the world health assembly, and some regional treaty bodies recognize palliative care and pain relief as elements of the right to health. ( ) governments have a minimum core obligation to protect this right with immediate effect, even in the face of a disastrous humanitarian emergency such as the covid- pandemic. global health experts describe lack of access to essential palliative care medicines as a critical public health issues, and opioids, in particular morphine, as essential for the relief of severe health-related suffering. the additional covid- burden of health-related suffering only underscores the government obligation to take a balanced approach to the regulation of internationally controlled substances, and to make strategic interventions, in partnership with clinical associations, to ensure the availability, accessibility, and affordability of essential medicines for primary, intensive, and palliative care. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. geneva: world health organization united nations office on drugs and crime: single convention on narcotic drugs new york: united nations alleviating the access abyss in palliative care and pain relief-an imperative of universal health coverage: the lancet commission report the international bank for reconstruction and development quantifying the adequacy of opioid analgesic consumption globally: an updated method and early findings access to pain treatment as a human right medicine shortage looms over coronavirus-hit europe ensuring and restoring balance on access to controlled substances for medical and scientific purposes: joint statement from palliative care organizations availability of internationally controlled essential medicines in the covid- pandemic pettus availability of internationally controlled drugs: ensuring adequate access for medical and scientific purposes the international narcotics control board calls on governments to ensure continued access to controlled medicines for pain relief and palliative care and for mental health and neurological conditions during the covid- pandemic eu authorities agree new measures to support availability of medicines used in the covid- pandemic characteristics, symptom management and outcomes of patients with covid- referred for hospital palliative care exclusive: opioid supply crunch for u.s. coronavirus patients prompts appeal to relax limits hospitals urgently call for more european collaboration to prevent drug shortages available at availability of internationally controlled essential medicines in the covid- pandemic pettus business insider. doctors are asking states to use their lethal-injection drugs to treat patients with covid- coronavirus: 'local shortages' of intensive care drugs key: cord- - knig g authors: thacker, s.b.; sencer, d.j. title: centers for disease control date: - - journal: international encyclopedia of public health doi: . /b - - . - sha: doc_id: cord_uid: knig g emerging from a small, wartime government program with a regional focus on malaria in , the centers for disease control and prevention (cdc) has become a global public health agency that addresses the entire scope of public health, with over employees and contractors in nearly occupations. the cdc's expertise has expanded in direct correlation with the expanding view of public health needs: it is recognized globally for its ability to respond to urgent threat related to disease epidemics and the health consequences of disaster and war. cdc programs have contributed significantly to the eradication and reduction of diseases such as smallpox, polio, and guinea worm, as well as the control of health problems such as human immunodeficiency virus (hiv), childhood lead poisoning, breast and cervical cancer, diabetes, violence, and unintentional injuries. cdc contributions in applied epidemiology, public health surveillance, risk factor reduction, and environmental risk assessment also have been critical to the practice of public health in the united states and around the world. the emerging concerns of the new century – genomics, globalization, the built environment, information technology, global warming, emerging infections, violence, and so forth – will require not only the traditional disciplines but also new expertise and new global partners, both public and private. the history of the centers for disease control and prevention (cdc) began in with the establishment of the malaria control in war areas (mcwa), under the u.s. public health service (phs). the u.s. military had suffered severely from malaria during world war i, and although the reported incidence had dropped during the s, a cyclical -to -year pattern of disease raised concern. because the disease had been endemic in the southern united states, concern was heightened because military bases and more than essential war establishment facilities were located there. mcwa was the actualization of the vision of joseph w. mountin, md ( - , an assistant surgeon general in the phs and director of the bureau of state services, who reported to the surgeon general of the phs. the headquarters was located in atlanta, georgia, with close associations with state health departments, puerto rico, and the virgin islands, as well as laboratory facilities and field stations in multiple states that worked with all the affected states and territories. although the phs provided leadership for the new program, much of the expertise in malaria had been recruited by the military; therefore, substantial training became an essential component of the multifaceted approach taken by mcwa. physicians assessed clinical malaria and parasitologists managed the laboratories; however, mosquito control was the emphasis, and engineers and entomologists dominated mcwa. field staff from the recently terminated works progress administration were recruited to continue their work of draining malaria breeding grounds and larviciding with diesel oil and insecticides (beginning with paris green but adding ddt in , which substantially changed the approach to malaria control). state laboratory staff were trained to diagnose malaria by using the most effective techniques. the program's scope expanded to the civilian population and to other vector-borne diseases such as dengue and typhus. mwca laboratory workers also responded to requests from states for assistance in epidemic investigations, a role previously left to the national institute for health (nih) (now the national institutes of health). the mcwa program was regarded as highly successful, and even before the end of the war, mountin and his staff were considering the future. with the support of surgeon general thomas parran, jr. (surgeon general, - ) , the communicable disease center was established on july , . at the time, cdc had employees and a budget of $ . million ( table ) . the legislation that created the cdc explicitly charged the new agency with responsibility for dealing directly with state health departments in the control of communicable diseases. this role was solidified in when the association of state health officials and the american public health association agreed that cdc take the lead in defining what diseases were of highest priority and should be reportable. the key figure in this expansion of the cdc role was alexander d. langmuir, md ( - ) , who was brought to the cdc in as chief of the epidemiology division. langmuir brought experience as a member of the armed forces epidemiology board, as a practicing epidemiologist at both the local and state health departments in new york, and as a professor teaching at the johns hopkins school of hygiene and public health. he also brought vision and a strong personality that helped bring the role of the epidemiologist to prominence at the cdc and in public health practice throughout the country. the cdc expanded during those first years to include field stations in missouri, colorado, and texas, and to conduct special studies in other states. influential events and decisions in the s, however, truly established the cdc as an agency with national recognition, and mountin and langmuir were forces behind the majority of these changes. in , the cold war set the tone in international affairs, and the korean conflict fueled a concern about the pointed use of biologic weapons there and in the united states. langmuir successfully championed the concept of the epidemic intelligence service (eis), which would respond to disease outbreaks as part of a program of biologic warfare defense. the eis program was established in with the recruitment of men, including physicians and a sanitary engineer. this first class became the disease detectives, the symbol of applied epidemiology practiced at the cdc and the core of epidemiologists who would come to lead the agency in future years. by , more than men and women had graduated from the program, all with different backgrounds and experiences. together they have conducted more than investigations, not only in infectious disease but also in chronic diseases, injury, and the many other health areas that the cdc has engaged in during subsequent years ( table ) . eis alumni have become public health leaders at the cdc, throughout the united states, and around the world. the event that first brought national attention to the cdc came with poliomyelitis, the crippling childhood disease. in , more than cases were reported in the united states, and polio was the leading infectious cause of childhood death in the country. in , the university of michigan conducted a national randomized trial of the killed virus vaccine developed by jonas salk on more than u.s. school children during a year when more than cases had been reported. the positive results were announced on april , , the tenth anniversary of the death of franklin delano roosevelt, and the response was dramatic. within hours, surgeon general leonard a. scheele (surgeon general, - ) announced a national vaccination program. unfortunately, however, on april of that year, a baby was reported to have contracted polio days after vaccination; this proved to be the index case of an epidemic of vaccine-associated polio that led to a decision to shut down the program. four days later, langmuir was directed to establish the national polio surveillance unit he had been advocating, and he immediately directed the eis officers to focus their efforts on this national emergency. within less than a week, daily reports were produced by the epidemiologists in the unit. rapid investigation in the field and in the laboratory clearly implicated one of the five vaccine manufacturers as the source of the epidemic (although a second manufacturer might have had problems as well). weeks later surgeon general scheele was able to announce that the problem had been identified, safety standards had been instituted, and the vaccine was now safe to distribute. the cdc's role had been critical, and the importance of public health surveillance and of the eis was recognized. national surveillance for asian influenza in - , together with the work of cdc epidemiologists and laboratory staff, cemented the national role of the agency in disease control. the second major event of the decade was less obvious to the public -the transfer of the phs venereal disease division (vdd) to the cdc in . at the time the vdd had a larger budget than the cdc and certainly a longer history. however, the effect was more important than the budget. the vdd brought with it a grant program that distributed money to states and a program management function -the public health advisor. the grant program enhanced the agency's connection and collaboration with state health departments, and the public health advisor became the primary nonscientific manager of cdc programs and of the agency itself. another program that came to the cdc with the vdd -unfortunately one that was highly negative -was the tuskegee study of the complications of syphilis, which had begun in table centers for disease control and prevention (cdc) timeline among black men in rural alabama. although the gravity of this study was little recognized in , the matter became public in and led to a formal apology by president william j. clinton in . the cdc moved into new facilities adjacent to the emory university campus in . at the time, the cdc had more than employees and a budget of $ million. the expansion of the agency mission was equally substantial. by the end of the decade, the budget had reached $ million, and the cdc encompassed programs in immunization, hospital infection control, tuberculosis, and environmental health. the journal morbidity and mortality weekly report (mmwr) was brought from the national office of vital statistics to the cdc by langmuir in . the publication rapidly became the agency's premier publication and the avenue for publishing concise, science-based articles of current events of public health interest such as epidemics, as well as current data regarding disease occurrence and death. the cdc increasingly was recognized for its responsiveness to epidemics of infectious diseases, including drug resistance among hospitalized patients, salmonella in commercially produced chicken, polio associated with oral vaccine, and for its international work in smallpox eradication and disaster assistance. the cdc also maintained its lead federal role in disaster assistance, and staff studied the immediate and long-term health effects of such disasters as hurricane camille in . possibly, the most important event for the cdc during that decade, however, occurred in geneva at the world health assembly in when the world health organization (who), under the joint leadership of the united states and the united soviet socialists republic, endorsed the plan to eradicate smallpox. this political will, together with improved technology (e.g., the jet injector gun to deliver vaccine), was the impetus for an extraordinary global program. donald a. henderson, md (eis ) , was transferred to who to help lead the international effort. a global mass-vaccination strategy was undertaken, and active surveillance documented rapid decreases in illness and death. however, the program began to founder and eradication appeared out of reach. william h. foege, md, another eis alumnus ( ) , demonstrated in west africa that an active surveillance and containment strategy was an effective complement to the mass-vaccination strategy. in , these efforts resulted in the first successful eradication of a human disease in history, years after the last case of wild-strain smallpox in somalia. the cdc's global role in disease control was founded on its active role in smallpox eradication. two future cdc directors, foege and jeffrey p. koplan, md, who had served as an eis officer ( ) in the program, and dozens of future public health leaders and hundreds of public health workers domestically and internationally were trained in this effort. the critical role of epidemiology and public health surveillance was now recognized by a much broader audience, and the importance of international, cross-disciplinary, and cross-cultural collaboration was appreciable. the s brought further change to the cdc and initiated its programmatic expansion to broader areas of public health, including environmental health, additional chronic diseases, occupational safety and health, and injury prevention and control. the cdc's expansion in part came from the transfer of programs in these areas, notably the national institute for occupational safety and health, which was transferred in . other expansions resulted from calls for assistance from states or other federal agencies. childhood lead poisoning was being reported as a result of industrial pollution or from parents bringing lead home on their work clothing. vinyl chloride-associated liver cancer was demonstrated in population studies. investigating the short-and long-term effects of exposure to radiation after the incident at the nuclear reactor at three mile island in pennsylvania was initiated at the end of the decade. a collaborative study with the national cancer institute was conducted to investigate the association of cancer with both oral contraception and estrogen therapy. all these changes provided background for major organizational changes that occurred at the end of the decade. meanwhile, the cdc continued to have active engagement in infectious diseases. the identification of salmonella among pet turtles altered that industry, as did discovery of the contamination of commercial intravenous preparations with bacteria and the subsequent documentation of a nationwide epidemic leading to a product recall. the study of the efficacy of nosocomial infection control (senic) confirmed the effectiveness of hospital infection-control practices. internationally, the cdc was involved in documenting the threat of hemorrhagic fever viruses -first the marburg virus in germany, and subsequently the lassa and ebola viruses in sierra leone, zaire, and sudan. two events in , the agency's th year, however, might have had the most enduring impact. in spring , an outbreak of influenza at a military base in new jersey was identified as being caused by a new strain of influenza a, a strain that was quite different than the strain (a/hong kong/h n ) circulating since . the new strain (h sw n ) had demonstrated pathogenicity among humans and its ability to be transmitted person to person, circumstances that were believed to always lead to pandemics. as important, this strain was believed to be closely related to the influenza virus that had led to a pandemic in - , which had killed u.s. residents and an estimated million persons globally, affecting particularly young adults. with the cdc in the lead, scientific experts were engaged during the following months to study the problem. they ultimately recommended the policy that led to the national influenza immunization program, which targeted the entire u.s. population. vigorous efforts by the president, congress, the vaccine manufacturers, and the public health system enabled the program to begin in october . within weeks, the cdc's national surveillance program, in collaboration with the states, uncovered an increased reporting of guillain-barré syndrome among persons who had been vaccinated during the program. subsequently, the program was suspended and no epidemic occurred. an association was confirmed in a national case-control study conducted by the agency; the agency was criticized harshly, and other vaccination programs were called into question. in early august , a report from pennsylvania of possible swine influenza led to an investigation of fatal pneumonias among veterans and their families who had attended a statewide convention of the american legion in philadelphia during the third week of july. the legionnaires' disease epidemic eventually brought eis officers and many other staff to pennsylvania and stayed on the front pages for weeks during this bicentennial year. by the end of august, the field team had identified more than cases -laboratory staff had ruled out all known human pathogens -and together they had investigated multiple leads in an effort to determine a toxin. terrorism was considered, and external experts were called in. however, the strongest association identified was with the hotel that had housed the conventioneers and had hosted major business and social activities. the team returned to atlanta without a definitive answer, and in the context of the mounting criticism around the swine influenza program, continued its work. finally, in december, joseph e. mcdade, phd ( -) , in the cdc's rickettsial diseases laboratory, identified the gram-negative rod that proved to be the bacterium that caused the epidemic. legionella pneumophila became the first new human bacterial pathogen identified in decades, and mcdade and his coworkers linked it to two previously unsolved epidemics in and . the s was dominated by the epidemic caused by hiv, but the cdc was also to undergo another major organizational change that reflected a new direction for the agency. however, the decade opened with two investigations of a more traditional nature that brought national visibility to the agency and brought lawyers into the public health policy arena in a dramatic fashion. in the summer of , a report from arizona in the mmwr linked reye's syndrome with using aspirin. that report was followed by similar data from ohio and michigan, which led the cdc to conclude that the association might be real. the aspirin industry aggressively attacked, but the cdc held firm and recommended that aspirin be avoided for children with chickenpox and during influenza epidemics. surgeon general julius b. richmond ( - ) soon followed with a similar recommendation. the struggle with industry did not stop, but national data demonstrated a noticeable decrease in reye's syndrome during the next few years. meanwhile, the cdc was responding rapidly to the national epidemic of toxic shock syndrome among women. within months, the disease was linked to use of tampons and by the end of , specifically to the use of the rely tampon, made by proctor and gamble. after the manufacturer voluntarily withdrew the product, the epidemic abated dramatically. major change was continuing to happen in public health, and the cdc was involved in measuring the health effects of the radiation release from the nuclear reactor at three mile island in pennsylvania, the toxic effects of dioxin at love canal (new york) and among vietnam war veterans, and the volcanic eruption at mount saint helens (washington state). the chemical release in bhopal, india, and the investigation of toxic oil syndrome in spain reflected the international recognition of the cdc in environmental health. in , the agency announced a reorganization to reflect these new public health concerns and environmental health, chronic diseases, occupational health, unintentional injuries, violence, and maternal and child health, while maintaining excellence in the prevention of infectious diseases. this reorganization enabled the programmatic growth for the next two decades. however, a brief report of an unusual pneumonia among five homosexual men in a june issue of the mmwr changed health practice at the cdc and around the world. the pandemic of acquired immunodeficiency syndrome (aids) caused by hiv crept slowly into public consciousness, but by the end of the decade, no health problem in the world generated more interest and controversy. the cdc was at the center of both. during those years, more than a third of the agency's budget was directed toward researching this illness and the virus that causes it. the agency's budget grew to approximately $ billion by the end of the decade. its constituency grew with the budget, and both domestic and global partnerships expanded considerably. at the cdc, behavioral and social scientists rapidly developed an important niche, and the move of the national center for health statistics in to what was now the centers for disease control brought a wealth of data and enhanced the role of the statistician at the cdc and in public health practice. the last decade of the twentieth century was marked by expansion of programs and budget in the newly defined priority areas at cdc, especially chronic diseases. in , the cdc's name changed to reflect its broadened mission, and it became the centers for disease control and prevention. by , the cdc budget had exceeded $ billion. the chronic disease budget was $ million that year and was dedicated primarily to development of programs for preventing breast and cervical cancer. in addition, routine data collection was being established at the state level through the behavioral risk factor surveillance system, and state-based cancer registries were implemented to provide a data baseline for defining problems and evaluating program effectiveness. emerging infections (e.g., escherichia coli o :h , the hantavirus, and west nile virus) and reemerging infections (e.g., antibiotic and drug resistance for bacteria, tuberculosis, and malaria), together with an increasing number of effective vaccines, underscored the continuing importance of infectious diseases. by the end of the century, ten major programs (then termed centers, institute, and offices) were located at the cdc, the last focusing on unintentional injuries and violence. an th center was created a year later. global health programs related to hiv and other worldwide public health concerns continued to grow. by , a global network of more than field epidemiology training programs modeled on the eis was established. on any give day, the cdc had more than persons stationed internationally on long-term assignments. nationally and globally, the science and services of the cdc were in great demand. the mmwr was available on the internet and in received . million hits. additional notable public health events marked the early years of the twenty-first century -the terrorist attacks on the world trade center and the pentagon (and the downing of a fourth airliner in a field in pennsylvania) and the deliberate anthrax poisonings in . those two major events were followed in by hurricanes katrina and rita occurring in rapid succession. all of these crises had an extraordinary effect on the country and on the cdc. the emergence of a major change in influenza made real the threat of another pandemic, and this also played out markedly at the agency. two important themes emerged from these events. first, the nation and the cdc needed to adapt their approach to public health emergencies. although the traditional approaches worked, improvement was needed. the impact on the public health system was significant and long-lasting. second, the cdc received substantial additional funding, most of which was provided to traditional partners in state and local health departments to strengthen public health system infrastructures and to adapt to new demands. more than staff are now employed at the cdc, and the budget in exceeded $ billion, $ billion of which was shared with state and local governmental partners and with nonprofit organizations. the new era stimulated a major examination of the agency and its future, and in led to a major reorganization that focused both on adaptation and response to crises but also greater collaboration across the major programs. the intent was to focus on major public health goals and not necessarily specific disease or injury programs, except in support of these goals. as with the reorganization two decades earlier, this process will take years to finalize and to assess the impact. beginning with a regional focus on a single parasite, the cdc has become the premier public health agency in the world, and its expertise has expanded in direct correlation with the expanding view of public health needs. the emerging concerns of the new century -genomics, globalization, the built environment, information technology, global warming, emerging infections, violence, and so onwill require not only the traditional disciplines but also new expertise and new global partners, both public and private. what will remain the same at the cdc is the dedication to its mission in global public health and its adherence to the core values of accountability, integrity, and respect. the new century has already proven challenging and exciting, a situation that cdc anticipates eagerly. ohio) and provides epidemiologic assistance to canada during the winnipeg flood -epidemic intelligence service (eis) established -cdc reports first case of bat rabies in united states -cdc establishes polio surveillance unit during national investigation of contaminated vaccine -asian influenza pandemic; venereal disease division (vdd) transferred to cdc -first assistance to southeast asia in response to cholera and smallpox epidemics -permanent facilities open on property adjacent to emory university campus; tuberculosis program transferred to cdc -morbidity and mortality weekly report (mmwr) moves to cdc; cooperative cholesterol standardization program established -foreign quarantine service joins cdc control; smallpox eradicated in west africa -national institute for occupational safety and health moves to cdc -mmwr reports that lead emissions in residential area are a public health threat -first field epidemiology training program established in canada -global smallpox eradication achieved (eradication certified by the world health organization in ) -first outbreak of multidrug-resistant tuberculosis (mississippi) reported -publication of healthy people established measurable public health goals for the united states for -agency for toxic substances and disease registry established; mmwr publishes first report of toxic shock syndrome associated with tampon use -mmwr publishes first report of fatal disease eventually called acquired immunodeficiency syndrome (aids) -cdc reorganized with new centers for infectious diseases cdc established violence epidemiology branch -office on smoking and health moves to cdc -national center for health statistics moves to cdc -national center for chronic disease prevention and health promotion established -phs recommends that all women of childbearing age consume mg of folic acid daily to reduce risk for spina bifida and anencephaly -national center for injury prevention and control established -cdc's prevention mission is recognized, and it becomes the centers for disease control and prevention -polio elimination certified in the americas -cdc reports measurable levels of serum cotinine in blood of % of nonsmokers in united states -cdc participates in presidential apology for the tuskegee study of syphilis treatment among black men -cdc's laboratory response network established -national center for birth defects and developmental disabilities established -director's emergency operations center opened -cdc provides global assistance for surveillance and clinical and laboratory evaluation regarding severe acute respiratory syndrome (sars) cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective-david satcher cdc's th anniversary: director's perspective sentinel for health: a history of the centers for disease control legionnaire's disease: description of an epidemic of pneumonia current cdc efforts to prevent and control human immunodeficiency virus infections and aids in the united states through information and education the cutter incident: poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of . i: background. ii: relationship of poliomyelitis to cutter vaccine. iii: comparison of the clinical character of vaccinated and contact cases occurring after use of high rate lots of cutter vaccine department of health and human services reflections on the swine flu vaccination program the epidemic intelligence service of the centers for disease control and prevention: years of training and service in applied epidemiology a tribute to alexander d partnerships in international applied epidemiology training and service key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of 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astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -q eiyp e authors: o’connell, mary beth; samman, leah; bailey, teresa; king, larissa; wellman, gregory s. title: attitudes of michigan female college students about pharmacists prescribing birth control in a community pharmacy date: - - journal: pharmacy (basel) doi: . /pharmacy sha: doc_id: cord_uid: q eiyp e in the united states, the overall unintended pregnancy rate is about %. women between – years old account for % of the unintended pregnancies. continuous birth control use is related to decreasing unintended pregnancies. therefore, we assessed female college students’ opinions about pharmacists prescribing birth control in a community pharmacy using an intersectionality framework. a survey with items about provider attributes, pharmacy services use and evaluation, advantages and barriers of pharmacists prescribing birth control, sexual and reproductive history, and demographics was distributed by survey link and qr code. recruitment was done by investigators and students (snowballing technique) via emails, social media posts, and direct student contact. respondents (n = ) were . ± . years old, % white, % healthcare students, % student pharmacists, % sexually active, % with at least one episode of unprotected intercourse within a year, and % never using condoms. forty-six percent of students were extremely likely and % moderately likely to get birth control from a pharmacist because it would be easier to adhere to birth control, could prevent unintended pregnancies, would be more convenient, and require less time. concerns included the lack of pap screenings and prescriptions written for the wrong birth control. within most student characteristics or attitudes assessed, at least % of the students would use this service. based on student opinions, female college students would use pharmacists prescribing birth control services. unintended pregnancies are recognized as individual, family, and societal concerns. forty-five percent of pregnancies are unintended in the united states with higher prevalence in those - years old, with low income, and/or from minority groups [ ] . the cost of unintended pregnancies was estimated to be $ . billion dollars per year [ ] . of the unintended pregnancies, % of the mothers give birth, % have an elective abortion, and % have a miscarriage [ ] . prior to an abortion, % of the women were not using contraception and % had wanted to use birth control but experienced barriers to obtaining it [ ] . about million women of childbearing age live in areas without federally funded facilities that prescribe birth control [ ] ; however, many of these areas would have a community pharmacy. other barriers include cost and insurance coverage of contraception, payment of aged - are particularly important since they have the highest prevalence of unintended pregnancy in the united states [ ] . therefore, the purpose of this study was to determine female college students' opinions about and willingness to use pharmacists for obtaining hormonal contraception in a community pharmacy across a wide range of student characteristics, attitudes, and health service opinions. investigators developed a survey that was distributed to a convenience sample. we chose an intersectionality approach by querying about characteristics, attitudes, sexual behaviors, and health services use and opinions ( figure ) that overlap to influence a woman's health decision making. the research project was deemed exempt via category criteria (because identity of the students would not be ascertained from the data and no criminal or civil liability or student damage could be determined from results) by two university institutional review boards. an assent information form was included as the first question of the survey. viewpoints of women aged - are particularly important since they have the highest prevalence of unintended pregnancy in the united states [ ] . therefore, the purpose of this study was to determine female college students' opinions about and willingness to use pharmacists for obtaining hormonal contraception in a community pharmacy across a wide range of student characteristics, attitudes, and health service opinions. investigators developed a survey that was distributed to a convenience sample. we chose an intersectionality approach by querying about characteristics, attitudes, sexual behaviors, and health services use and opinions ( figure ) that overlap to influence a woman's health decision making. the research project was deemed exempt via category criteria (because identity of the students would not be ascertained from the data and no criminal or civil liability or student damage could be determined from results) by two university institutional review boards. an assent information form was included as the first question of the survey. female college students of any age from any college in any curriculum were eligible to participate. students selecting intersex as their gender were included but male students were excluded. surveys needed to have at least one other item besides gender answered to be included. surveys from students residing or attending college outside of michigan were excluded. recruitment was primarily conducted at two universities and two community colleges. these educational institutes were chosen to gather student opinion throughout the whole state and provide metro, urban, and rural geographical variability. one university was in detroit, one community college in a detroit suburb, one university on the west side of the state, and one community college in the upper peninsula. a validated survey related to our project was not available. literature was reviewed related to over-the-counter birth control, emergency contraception, pharmacists prescribing contraception, female college students of any age from any college in any curriculum were eligible to participate. students selecting intersex as their gender were included but male students were excluded. surveys needed to have at least one other item besides gender answered to be included. surveys from students residing or attending college outside of michigan were excluded. recruitment was primarily conducted at two universities and two community colleges. these educational institutes were chosen to gather student opinion throughout the whole state and provide metro, urban, and rural geographical variability. one university was in detroit, one community college in a detroit suburb, one university on the west side of the state, and one community college in the upper peninsula. a validated survey related to our project was not available. literature was reviewed related to over-the-counter birth control, emergency contraception, pharmacists prescribing contraception, obstetrics and gynecology practice/provider patient preferences, pharmacy clinical services in community pharmacies, and position statements related to these topics to create our survey. survey items were developed based on published non-validated survey items or newly created to assess this study's purposes. a -item survey (appendix a) was initially developed with two additional items (i.e., college attending and current city of residence) added after the study started. the survey topics (in survey order) were assent ( item), health care experience attributes of gynecologists or clinic providers and future pharmacists prescribing birth control ( items with sub-items each), pharmacy services and evaluation ( items), pharmacist prescribing birth control advantages and barriers ( items), demographics ( items), and sexual and reproductive history ( items) . for the health care experience attributes section, students were presented with two scenarios where she obtained a birth control prescription from a gynecologist or primary care provider and one from a community pharmacist with the ability to prescribe birth control. students were then asked to rate their perception of nine provider attributes for both scenario providers on a scale of one (lowest) to ten (highest). students could skip answering any of the items. no personal identification information was collected. a second survey was created for the raffle of two $ gift certificates. the survey platform was qualtrics. the survey was distributed to ten students from the two universities for review of item clarity and understanding, and survey completion time. suggestions were incorporated into the final survey. the qualtrics survey url link was used and later the survey qr code was added to advertisements. one university posted an advertisement two times a week in a daily newsletter sent out to every student over a span of six weeks. they also sent a mass email containing the survey link to all college of pharmacy students at this university. at the other university, a study notice or flyer was posted in student news feeds, research recruitment webpage, campus housing, facebook pages and other social media. both universities sent emails to female student organizations asking for them to email or post the advertisements. some in class pharmacy and other program announcements were made. students were also asked to participate while dining in the university commons area. one community college sent out broadcast emails to the whole student population. the other community college sent advertisement to one curriculum, i.e., nursing, for which a student mass email list existed. students participated in snowballing techniques by sending emails to their female college friends or posting on their own social media sites. recruitment occurred from april to february . target goal of completed surveys was not achieved. descriptive statistics with inferential statistics, chi square, and ordered logistic regression were used to analyze the data. for chi square analyses, five-point likert scales were collapsed to three-point likert scales. for likelihood of obtaining birth control from a community pharmacist, those stating no birth control use were excluded from crosstab analysis. extremely and moderately likely responses were collapsed to likely, not sure responses remained, and not very likely and not likely at all responses were collapsed to not likely. these analyses were done with ibm spss statistics version . (ibm corp., armonk, ny, usa). for the ordered logistic regression, the full five-point likert scale and the students not using birth control were included. the pharmacist attributes were on a scale of one being low to ten being high. this analysis was run on of the respondents using listwise deletion. the ologit module in stata . was used for this analysis. a p-value of less than or equal to . was considered significant. because of the survey distribution techniques, total sampling cannot be quantitated and therefore a response rate cannot be calculated. the survey was opened by students with ( . %) surveys included in the analysis. the reasons for survey deletions were surveys had no responses, were started by male students, had no answers except for gender ( intersex, women), were completed by female students attending non-michigan colleges, and were completed by female students living outside of michigan. the average age and standard deviation of the participants was . ± . years (range - years old). the student participant characteristics and associated likelihood to obtain birth control from a pharmacist are in table . overall, female college students stated they would obtain birth control from a pharmacist with most students stating they were extremely likely ( . %) to do so, followed by . % moderately likely, . % unsure, . % not very likely, and . % not likely at all. the mean response and standard deviation was . ± . [six-point scale with one (extremely likely) to six (never)]. students had positive comments about pharmacists prescribing birth control but also some concerns. survey advantages and concerns prompted responses (survey items) are listed in table . the mean number of prompted and other stated advantages was . ± . with most students listing five ( . %) or six ( . %) advantages. additional advantages included a pap or pregnancy test not needed ( ), insurance coverage checked ( ), birth control switching easier ( ), less stress ( ), blood pressure checked ( ), fewer appointments ( ), pharmacists more knowledgeable ( ), pharmacists more available for questions ( ), easier to get in an emergency ( ), easier to get refill ( ), and easier to get answers ( ). five students ( . %) listed no advantages. the mean number of prompted and other stated concerns were . ± . with most students listing one ( . %) or two ( . %) concerns (table ) . additional concerns included no medical chart access ( ) , pharmacists are overworked ( ) , relationship with physician needed ( ), lack of privacy ( ), less education than other providers ( ), pharmacists need more birth control education ( ), insufficient patient education provided ( ), lack of insurance coverage of visit ( ), should be pharmacist and doctor team ( ), limited birth control options to be prescribed ( ), pharmacist not able to resolve side effects ( ), pharmacist refusal to dispense ( ), non-hormonal options needed ( ), less professional environment ( ), lack of documentation ( ), and uncomfortable with pharmacists prescribing ( ). forty-eight students ( . %) listed no concerns. in table , the percent of students stating they were extremely or moderately likely to get their birth control from a pharmacist in a community pharmacy is displayed. only citizenship, degree program, health professional student status, and type of health professional student had significant differences within the category. sixty-seven to % of the various religious/spirituality groups would obtain birth control from a pharmacist. one third of the students would use birth control even if their religion opposed this medication. one percent stated because their religion did not support birth control, they would not use this medication. for . % of the students, they stated their religion only approved birth control if married, and . % stated permission to use birth control only after marriage and with partner approval. almost a third of the students ( . %) had no beliefs that would impact birth control use. some students ( . %) had religions that did not oppose birth control use. sexual practices of the students and likelihood of obtaining birth control from a pharmacist results are presented in table . many students ( . %) were in a sexually active relationship with a man and % with a woman. about % of the students stated they did not need birth control because they were not sexually active ( . %), or were incapable of getting pregnant ( . %), pregnant ( . %), or postmenopausal ( . %). nineteen students ( . %) practiced abstinence. prescription or provider administered birth control used by the students were % pills, . % iuds, . % implants, . % shots, . % emergency contraception, and . % rings. of those using hormonal birth control, most students had only one indication ( . %), with . % having two indications, and . % having three indications. the most common indication for birth control was contraception ( . %), followed by menstrual disorders ( . %), acne ( . %), and other ( . %). for the specific sexual practice variables, % to % of the female college students stated they were extremely or moderately likely to get their birth control from a pharmacist (table ) . only sexually active with a man (p = . ) and times of unprotected intercourse in the last year (p = . ) had significant differences within the variable. an ordinal regression on likelihood of obtaining birth control from a pharmacist did not yield a significant model for the variables age, age at first intercourse, number of sexual partners, or number of times intercourse per week (model significance p = . ). previous use and quality assessment of pharmacy services and their impact on opinions about obtaining birth control from a pharmacy are depicted in figure . having received a vaccination from a pharmacist (p = . ), having greater confidence in pharmacist prescription dispensing (p = . ) and counseling (p = . ), and believing pharmacists have more knowledge than their doctor, nurse practitioner, or physician assistant (p = . ) were associated with greater likelihood of getting their birth control from a pharmacist. most female college students thought that receiving birth control from their pharmacist would definitely ( . %) and probably ( . %) increase their birth control adherence, and would definitely ( . %) and probably ( . %) decrease unintended pregnancies. the greater the belief of an impact of pharmacists prescribing birth control on increasing birth control adherence (p = . ) and decreasing unintended pregnancies (p = . ), the greater the likelihood of receiving their birth control from a community pharmacist. students ranked personal attributes of a pharmacist in the . to . (out of ) range with some aspects of the pharmacy and getting birth control in that environment lower ( . to . ). the results are shown in table . using the nine attributes in table , an ordered logistic regression analysis was done with the variable female college students' likelihood of getting their birth control filled at a community pharmacy. the scale for likelihood of getting their birth control filled at a community pharmacy was reversed to aid in the interpretation of the odds ratios. the model was significant in describing pharmacist prescribed birth control (p < . ). the attributes trustworthiness (or . , ci . - . , p < . ), approachability (or . , ci . - . , p = . ) and visit expense (or . , ci . - . , p = . ) were statistically significant in predicting likelihood of using a pharmacist as a primary provider for contraceptives. the greater the perception of trustworthiness and approachability the students had for the pharmacist, the greater their likelihood to use the pharmacist as a primary provider for birth control. the lower the students perceived the pharmacy visit expense to be, the more likely they would use the pharmacist as a primary provider of birth control. this study provided evidence that women are interested in pharmacists prescribing birth control. seventy-three percent of michigan female college students felt they were likely to obtain their birth control from a community pharmacist. they felt the community pharmacist visit for birth control would be more convenient, save time, and make obtaining birth control easier. they also felt this pharmacy service would help them continue and be more adherent to their birth control, and prevent unintended pregnancies. the predominant concerns the students had were not getting regular pap smears and screenings and potentially being prescribed the wrong birth control. these advantages and concerns are similar to other studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many women do not know that a pap smear and breast exam are not required for birth control use [ ] . in terms of targeting this service to select groups of college students, we found across all our student characteristics to have high support for obtaining birth control in a community pharmacy, making the service generalizable across diverse students. overall, at least % of the female college students across the various student characteristics stated they would use pharmacist birth control services, a measurement of acceptability. no statistically significant differences were seen between race and ethnic groups, religion/spirituality groups, residence while going to college, size of city living in during childhood, political party, type of college, student enrollment status, employment, and medical or medication insurance coverage. students from a foreign country (i.e., student visa or immigrant status) were more likely to use this service than american born and naturalized citizen students, which might be related to lack of a primary care provider, health insurance, and or transportation, or non-prescription access to contraception in their birth country. healthcare students were more likely than non-healthcare students to use a pharmacist for birth control, which might reflect greater understanding of pharmacist education and training and subsequent medication expertise. variability within the healthcare students existed with pharmacy students having the highest, medical students in the middle, and nursing students the lowest likelihood. these differences could be related to territoriality and profession-centrism regarding writing prescriptions for patient management [ ] . no differences existed within the various non-healthcare student disciplines, which could mean similar awareness of pharmacist skills and training. graduate/professional degree students were more likely to use their pharmacist for birth control than undergraduate and associate degree students, which could represent healthcare professional awareness, maturity, and independence in health decisions. we thought more risky sexuality practices might increase willingness to access birth control from a pharmacist, but variability in willingness to use this service did not vary by type of relationship, number of unprotected intercourse events per year, condom use, or worry about getting pregnant. our study is the first study with a diverse female college participant cohort to assess opinions about pharmacists prescribing birth control, a group which could use this service. we chose this cohort since college students are in the age groups with the highest unintended pregnancy rates [ ] . another college student study surveyed california student pharmacists and almost all the student pharmacists ( %) wanted to provide this service [ ] . they were not asked if they would use this service. these student pharmacists identified similar advantages as our college students about this pharmacy service. they had more concerns than our students such as the adequacy of pharmacist training, lack of a medical chart with concerns about patient data adequacy, and no incentive for pharmacists providing this service. by knowing more about pharmacy operations, student pharmacists could conceptualize this service differently than non-student pharmacists. teenagers and young adults also liked the privacy and convenience of the community pharmacist prescribed birth control [ , [ ] [ ] [ ] . they also had some similar concerns to the college students such as pharmacists' abilities, minimal patient specific data available, and limited birth control products. similar to our student cohort, analysis of public commentary to news articles from women and men in oregon supported pharmacists prescribing birth control, citing the service would be an asset and allow women to make reproductive health decisions [ ] . within the comments were similar concerns about incorporating this service into pharmacy operations, importance of physician patient relationship, safety issues, and insurance coverage. unintended pregnancy rates are higher in minority women and those living in poverty [ ] . in our study, many minority women ( - %) would get birth control from a pharmacist. also, in our study, % of the female college students with a public insurance plan would get their birth control at a pharmacy. how many of these students would use this service is unknown. in oregon, a state in which medicaid offers and pays for this service, % of women with this insurance benefit chose a pharmacy to obtain their new birth control prescriptions [ ] . in california, oregon, and new mexico, pharmacists offered birth control prescribing in rural and urban community pharmacies [ , ] , which would align with our female college students in rural, small, and urban residences willing to get birth control from a community pharmacist. past experiences with pharmacists influenced the female college students' likelihood of having a pharmacist prescribe birth control to them in a community pharmacy. using pharmacist birth control services in the future varied based on past pharmacy services used and confidence in pharmacists. having received a vaccination from a pharmacist and being more confident with pharmacists filling prescriptions and providing counseling were associated with a greater likelihood of obtaining birth control from a community pharmacist. about one third of the students ( . %) thought pharmacists knew more about birth control than primary care providers, which could be influenced by the high percentage of pharmacy students in the database. students' rankings of pharmacist attributes were very positive, at least . or higher on a -point scale. pharmacy attributes such as privacy, time, and cost were lower but still above . on a -point scale. pharmacist trustworthiness, approachability, and visit expenses significantly influenced their likelihood of using birth control pharmacists. in the opinion-based studies about pharmacist prescribing, women of all ages would use or support this service, but they do express concerns as well [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our research adds that prior use of pharmacy services and perceptions of pharmacist attributes influence these opinions. our study had a few limitations. the results represent one midwest state and only college student opinions, which might not represent other states or women of different ages. within all demographics, likelihood to use pharmacist birth control prescribing was high. the chance to win one of two $ gift cards was most likely not an incentive for survey completion. since the survey incentive was insignificant, respondent bias could exist with those more positive or opposed more likely to complete the survey. recruitment was challenging with only completed surveys after mass emailing, social media posts, and face to face recruiting at two major universities and two community colleges. difficulty recruiting for another public perception of pharmacists prescribing birth control also was noted in a study using social media to recruit women of any age [ ] . only . % of , social media advertisements on facebook were clicked on by viewers, and only . % of those clicks resulted in opening the survey. although the survey had good diversity with most demographic variables, race and ethnicity diversity were limited with % of the students being white and . % being hispanic. in michigan, % of the undergraduate college students are white, . % black, . % hispanic, . % asian, . % american indian or alaska native, . % of two or more races [ ] . we attempted targeting minority female college organizations and courses, but these strategies were not successful. our study represented the group with highest number of unintended pregnancies ( % were - years old) but other groups such as teenagers ( %) and those years and older ( %) were less represented. the survey was long because we had areas to explore. survey burden might explain the number of students that opened the survey without answering anything besides their gender or did not finish or skipped certain sections. our pre-study student sample stated the survey was doable. some but not all students might have been aware of pharmacists prescribing birth control prior to the study so different degrees of previous opinion development. many student pharmacists could have learned about this service in class or at national and state pharmacy meetings. learning about this service might have occurred in other curriculums. medical students might have discussed the american college of obstetrics and gynecology preference for over-the-counter birth control, which could have influenced their opinions on pharmacists prescribing contraception. little is known about the coverage of this new pharmacy service in other healthcare and non-healthcare (e.g., law, women's studies) student curriculums. at the initiation of this survey, this pharmacy service was not available in michigan. now one large pharmacy chain in michigan is offering this service through a collaborative practice agreement. the pharmacy chain started its service the month after our recruitment ended, which was the month covid- hit michigan. prior to covid- , minimal marketing had occurred and during covid- , pharmacists prescribing birth control was not the focus of pharmacy patient health care, so most likely this new pharmacy provider of birth control had little impact on students' opinions. many female college students in michigan are receptive to pharmacists prescribing birth control in a community pharmacy. they feel this service will make getting and adhering to birth control easier and decrease their worries about getting pregnant. their positive attitude results combined with reported prevention of unintended pregnancies and decreased health care costs outcomes in oregon [ ] support all states offering pharmacists prescribed birth control services. further research is needed to confirm women's and societal benefits and use of pharmacists prescribing birth control. the authors would like to thank travis blume, bay de noc community college, and mary miles, e.d, oakland community college health professions for recruiting at their colleges, and all students that helped with snowball recruitment strategies. the authors declare no conflict of interest. q recall a previous experience with, or imagine visiting, a gynecologist or clinic provider for a prescription for birth control. how would you rate each of the below topics related to that visit on a scale of being the lowest to being the highest? provider trustworthiness provider approachability provider respecting me provider knowledge of birth control birth control education received privacy ease of making an appointment time commitment-all time related to getting the birth control prescription, e.g., calling to get an appointment, waiting time to see provider, provider interaction, getting the prescription filled expense-all costs related to getting the birth control prescription, e.g., clinic visit, prescription cost, travel q imagine you want to begin taking birth control while away at college. you opt to go see a pharmacist at your community pharmacy, who now has the legal right to prescribe birth control. the pharmacist will ask you a series of questions and then determine which birth control would be best for you. the pharmacist would write your birth control prescription that could be filled that day in the pharmacy. how would you rate each of the below topics related to that visit with the pharmacist on a scale of being the lowest to being the highest? q thank you for participating in this study. we appreciate your time and responses. your responses will be very helpful to understand the needs of women in michigan and your opinions about a potential new women's health service-pharmacists prescribing birth control in a community pharmacy. would you like to enter the raffle for a chance of receiving one of the two $ gift cards? your name and email will not be connected with your survey responses. yes enter me in the draw do not enter me in the draw declines in unintended pregnancy in the united states american college of obstetricians and gynecologists committee on gynecologic practice. committee opinion: over-the-counter access to oral contraceptives contraceptive needs and services contraceptive practices, preferences, and barriers among abortion clients in north carolina lack of access = lack of power to decide american college of obstetricians and gynecologists committee on health care for underserved women healthy people . family planning. available online evidence summary: prevent unintended pregnancy cdc's / initiative impacts of four title v, section abstinence education programs: final report new research findings on programs to reduce teen pregnancy; the national campaign to prevent teen and unplanned pregnancy adolescent pregnancy and contraception american college of obstetricians and gynecologists committee on gynecologic practice: acog. committee opinion summary: over-the-counter access to oral contraceptives unprotected intercourse among women wanting to avoid pregnancy: attitudes, behaviors, and beliefs opening new doors to birth control state efforts to expand access to contraception in community pharmacies expanding adolescent access to hormonal contraception: an update on over-the-counter, pharmacist prescribing, and web-based telehealth approaches hormonal contraception prescribing by pharmacists: update association of pharmacist prescription of hormonal contraception with unintended pregnancies and medicaid costs youth perspectives on pharmacists' provision of birth control: findings from a focus group study older teen attitudes toward birth control access in pharmacies: a qualitative study adolescents' perceptions of contraception access through pharmacies public perception of pharmacist-prescribed self-administered non-emergency hormonal contraception: an analysis of online social discourse pharmacist outlooks on prescribing hormonal contraception following statewide score of practice expansion student pharmacist perspectives on providing pharmacy-access hormonal contraception services provider opinions regarding expanding access to hormonal contraception in pharmacies interprofessional education: a summary of reports and barriers t ; o recommendations pharmacist provision of hormonal contraception in the oregon medicaid population an evaluation of the implementation of pharmacist-prescribed hormonal contraceptives in california availability of pharmacist prescription of contraception in rural areas of oregon and new mexico college undergraduate enrollment snapshot this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -q wx k authors: resende, lucilene aparecida; aguiar-soares, rodrigo dian de oliveira; gama-ker, henrique; roatt, bruno mendes; de mendonça, ludmila zanandreis; alves, marina luiza rodrigues; da silveira-lemos, denise; corrêa-oliveira, rodrigo; martins-filho, olindo assis; araújo, márcio sobreira silva; fujiwara, ricardo toshio; gontijo, nelder figueiredo; reis, alexandre barbosa; giunchetti, rodolfo cordeiro title: impact of lbsapsal vaccine in canine immunological and parasitological features before and after leishmania chagasi-challenge date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: q wx k dogs represent the most important domestic reservoir of l. chagasi (syn. l. infantum). a vaccine against canine visceral leishmaniasis (cvl) would be an important tool for decreasing the anxiety related to possible l. chagasi infection and for controlling human visceral leishmaniasis (vl). because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of leishmania parasites, their inclusion in an anti-leishmania vaccine has been investigated in past decades. we investigated the immunogenicity of the “lbsapsal” vaccine (l. braziliensis antigens, saponin as adjuvant, and lutzomyia longipalpis salivary gland extract) in dogs at baseline (t( )), during the post-vaccination protocol (t( rd)) and after early (t( )) and late (t( )) times following l. chagasi-challenge. our major data indicated that immunization with “lbsapsal” is able to induce biomarkers characterized by enhanced amounts of type i (tumor necrosis factor [tnf]-α, interleukin [il]- , interferon [ifn]-γ) cytokines and reduction in type ii cytokines (il- and tgf-β), even after experimental challenge. the establishment of a prominent pro-inflammatory immune response after “lbsapsal” immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism ( . %) and indicating long-lasting protection against l. chagasi infection. in conclusion, these results confirmed the hypothesis that the “lbsapsal” vaccination is a potential tool to control the leishmania chagasi infection. leishmania infantum (syn. l. chagasi) is an intracellular protozoon that cause a severe systemic disease named visceral leishmaniasis (vl) [ ] . it is widely distributed in the mediterranean basin, middle east, and south america. vl has an annual incidence of approximately , cases [ ] . brazil declared a total of , clinical vl cases between and , and this number accounts for % of all reported vl cases in the americas; however, it is subject to substantial under-reporting [ ] . different geographical regions of the globe where visceral leishmaniasis is endemic, present the dogs as main reservoir for the l. chagasi that play a relevant role in transmission of the parasite [ , ] . dogs are also excellent models for the study of vl because the natural history of the disease in dogs and in humans is similar [ ] , especially regarding parasite-host interaction, immune response, and the development of new vaccines [ ] . the natural history of canine vl (cvl) has been well-described, particularly in regard to the parasite load in different tissues and the immunopathological changes according to progression of clinical forms [ , , , , , , , , , ] . a vaccine against cvl would be an important tool in the control of vl and would decrease the anxiety associated with l. chagasi infection in humans [ ] . the induction of long-lasting cell-mediated immune response triggering high levels of protection against cvl is considered as prerequisite of an ideal vaccine for controlling the l. chagasi transmission. different vaccine candidates against cvl have been reported showing the ability to induce immunoprotective mechanisms [ , , , , , , , , ] . in this sense, it has been shown that leishmune (fucose manose ligand plus saponin as adjuvant; zoetis, são paulo, brazil) presented % protection in a phase iii study [ ] . leishmune provided protection associated with the ability to trigger early and persistent activation of neutrophils and monocytes, in addition to activation of t cd + and t cd + lymphocytes displaying high levels of ifn-γ in the cd + t-cell subset [ , ] . additionally, the leish-tec vaccine (hertape calier, juatuba, brazil; a antigen plus saponin as adjuvant) has also been shown to induce increased levels of ifn-γ in four out of seven dogs with l. chagasi-infected bone marrow [ ] . more recently, a vaccine composed of excreted/secreted antigens of l. infantum promastigotes, referred as liesap has been shown to induce increased levels of ifnγ and nitric oxide (no), thus supporting its leishmanicidal effect [ ] . the efficacy of the lie-sap vaccine was reported as % in a double-blind randomized study [ ] . liesap is commercially available as canileish (virbac, carros, france) and is able to induce a th profile and reduce the parasitic load of infected macrophages co-cultured with lymphocytes from immunized dogs [ ] . the lbsap (l. braziliensis crude antigens plus saponin as adjuvant) and "lbsapsal" (l. braziliensis crude antigens plus saponin as adjuvant and lutzomyia longipalpis saliva) vaccines against cvl has also shown to induce a strong immune-mediated response. the lbsap and "lbsapsal" presented higher levels of circulating t-cell subsets (cd + , cd + ) and b lymphocytes (cd + ), as well as leishmania-specific cd + and cd + t cells [ , , , ] . furthermore, lbsap-vaccinated dogs presented high ifn-γ and low interleukin (il)- and transforming growth factor (tgf)-β expression in the spleen, resulting in a significant reduction of parasite load in this tissue [ ] . additionally, lbsap has been shown to induce a prominent pro-inflammatory immune response characterized by increased levels of both il- and ifn-γ and decreased levels of tgf-β by peripheral blood mononuclear cells (pbmcs), which were associated with parasite control in dogs [ ] . the incorporation of salivary proteins of sand flies have been widely used in experimental challenge studies, and the results suggest that this could be a good strategy to protect against leishmania infection [ , , , , , , , , , ] . previous studies of dogs using the "lbsapsal" vaccine displayed higher counts of circulating and leishmania-specific cd + t cells in addition to high nitric oxide (no) production [ ] and reduction of splenic parasite load [ ] . considering the inclusion of saliva from sand flies as a promising compound in vaccines against leishmania infection, we describe additional biomarkers induced by the "lbsap-sal" vaccine in dogs. we considered the previous vaccine protocol (t ), post-vaccine protocol (t rd ), and early (t ) and late (t ) periods of the l. chagasi-challenge. all research involving dogs was carried out according to the regulations of the brazilian society of science in animal research, adopted by the federal university of ouro preto ethics committee in animal experimentation, that approved the technical procedures using dogs under protocol number / . all dogs included in this study, were born and bred in the kennel facility at universidade federal de ouro preto in ouro preto, minas gerais, brazil. this kennel facility was established with male and female mongrel dogs, with negative serological and molecular/parasitological diagnosis for leishmania infection, donated by the local zoonosis control center from belo horizonte, minas gerais, brazil. the filial generations, comprising of offsprings resulting from the cross between the original mongrel dogs pairs were used further maintain the mongrel dog colony. in the present study, twenty mongrel dogs ( males and females), with seven months of age and negative results for indirect fluorescence immunoassay to anti-leishmania antibodies, were selected and received an anti-helmintic treatment and were submitted to polyvaccination protocols, including rabies (tecpar, curitiba-pr, brazil), leptospira, parvovirus, canine distemper, type ii adenovirus, parainfluenza and coronavirus (vanguard htlp / cv-l; pfizer animal health, new york, ny, usa). prior the experimental onset, the animals were maintained in quarantine in kennel runs ( m length x m width x m height) completely covered with stainless steel wire mesh to prevent the entry of sand flies. the kennel facilities were sprayed with pyrethroid insecticide, every three months, as a measure to control insect access. each run was installed with an infrared lamp heating ( watts) to ensure the thermal comfort of animals during the night and on cold days. all the dogs were housed without environmental enrichment (e.g. toys, exercise regimes, etc). the dogs were monitored twice a day, as routine inspection, during which the responsible veterinary was in charge of stimulating and playing with them to aiming to avoid behavior problems and minimize their suffering or distress throughout the study. all the experimental procedures described in this study, including this aspect of animal care was approved by federal university of ouro preto ethics committee in animal experimentation (protocol number / ). the animals were maintained with water and food ad libitum throughout the experiment time. the euthanasia of all dogs was performed under the supervision of a veterinarian physician, using barbiturate anesthetic (thiopental sodium, mg/kg, iv) followed by intravenous injection of saturated solution of potassium chloride. four groups of dogs involved in experiments were treated as follows: (i) "control" (c) group (n = ; subcutaneous injections of ml of sterile . % saline); (ii) "sal" group (n = ; subcutaneous injections of salivary lutzomyia longipalpis gland extract (sge; obtained as previously described- [ ] ) in ml of sterile . % saline); (iii) "lbsal" group (n = ; subcutaneous injections of μg of l. braziliensis promastigote antigen and sge in ml of sterile . % saline); and (iv) "lbsapsal" group (n = ; subcutaneous injections of μg of l. braziliensis promastigote antigen plus mg of saponin and sge in ml of sterile . % saline). all animals received three injections in the right flank at days intervals. the l. chagasi-experimental challenge in all dogs was performed after days of vaccination protocol, using intradermal promastigotes during the stationary phase of cultivation. the challenge was performed in the inner side of the left ear including five lutzomyia longipalpis salivary gland acini. all the analyzed dogs were euthanized days after l. chagasi-experimental challenge and the spleens were collected to evaluate parasite loads. the rationale for choosing such a long endpoint ( days after challenge) was based on the chronic course of the experimental canine visceral leishmaniasis after intradermal l. chagasi-challenge as previously reported [ ] . the vaccine was obtained as previously described [ ] . briefly, l. braziliensis (mhom/br/ / m ) promastigotes were obtained by in vitro culture in neal, novy, nicolle/liver infusion triptose media [ ] , and the parasite was fully disrupted by ultrasound treatment ( w, min, °c), aliquoted and stored at − °c. protein concentration was determined according to the method of lowry [ ] . the sge used in this study as the antigenic component of the vaccine was obtained from lutzomyia longipalpis females that were not fed, aged days, and dissected in slightly hypotonic unbuffered saline . %, as described in [ ] . after collection, the glands were disrupted in a sonicator for seconds and centrifuged at , g for min. the supernatant was collected and stored in a freezer at - °c until use. jugular vein was used for collecting ml of peripheral blood in sterile heparinized syringes. we analyze distinct times: baseline before vaccination (t ), days after third immunization dose (t rd ) as well as early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the pbmcs were isolated as previously described [ ] . briefly, the whole blood samples were added over ml of ficoll-hypaque (histopaque ; sigma, usa), centrifuged at g for min at room temperature, and the pbmcs were washed twice with rpmi ( g for min at room temperature). the pbmcs were resuspended in rpmi at cells/ml. the pbmcs cultures were performed in -well flat-bottom tissue culture plates (costar, cambridge, ma, usa). the in vitro assays were performed using μl of pbmcs ( . × cells/well) with μl of vaccine l. braziliensis-soluble antigen (vsa; μg/ml) or μl of soluble l. chagasi antigen (slca; μg/ml). the control cultures (cc; unstimulated) were analyzed using μl of rpmi in place of the antigenic stimulus. incubation was performed in a humidified % co atmosphere at °c for days, after which time the supernatants were collected and stored in a freezer at − °c for detection of cytokine and no. the quantification of cytokines was carried out by enzyme-linked immunosorbent assay (elisa) according to the manufacturer's instructions (r&d systems, minneapolis, mn, usa), as previously described [ ] . minimum cytokine sensitivity detection levels were pg/ml (il- ), pg/ml (tnf-α and ifn-γ), pg/ml (il- and il- ) and pg/ml (tgf-β). the analysis of il- (anti-canine il- /il- p , catalog number dy ), ifn-γ (anti-canine ifn-γ, catalog number dy b), tumor necrosis factor (tnf)-α (anti-canine tnf-α/tnfsf a immunoassay; catalog number dy ) and il- (anti-canine il- , catalog number dy ) cytokines were performed using duoset elisa. quantikine kit (mouse/rat/porcine/canine tgf-β immunoassay, catalog number mb b) (r&d systems, minneapolis, mn, usa) was used to measure tgf-β levels. the analysis of il- levels employed the capture antibody (monoclonal anti-canine il- antibody-catalog number mab ); the standard curve was obtained using recombinant canine il- (catalog number cl); and streptavidin (dy ; r&d systems/usa), biotinylated anti-canine il- antibody (catalog number: baf ) and substrate solution ( : mixture of h o and tetramethylbenzidine; product code - - ) were used. all experiments were performed according to the instructions of r&d systems using -well plates (corning incorporated, costar , washington, dc, usa). the microplate automatic reader (el ; biotek, winosski, vt, usa) was employed at a wavelength of nm. the measurement of no levels in supernatants of pbmcs cultures was performed by indirect method that quantify the nitrite concentration by griess reaction [ , ] . briefly, μl of griess reagent ( % sulfanylamide, . % naphthylethylene-diamide-dihydrochloride, and . % phosphoric acid; all from sigma, usa) was mixed with μl aliquot of cell-free culture supernatant. the microplate reader (biotek, el ) was used to analyze the absorbance at nm, after min of incubation at room temperature in the dark. the final nitrite concentration was determined based on a standard curve interpolation constructed by using sodium nitrite solutions in the range of - μm. the interference of nitrites already present in the culture medium was discounted; data were calculated by taking into account the blank, as control reaction, assayed by using the pbmcs cultures medium. the data were expressed as nitrite concentration (μm). the spleen specimens ( mm) were collected during necropsy and stored at - °c until use for dna extraction. the wizard™ genomic dna purification kit (promega, madison, wi, usa) was used to extract total genomic dna in mg of spleen following manufacturer's recommendations. primers that amplified a -bp fragment of a single copy of the dna polymerase gene of l. chagasi were used to analyze the spleen parasite burden by quantitative polymerase chain reaction (qpcr). for the q pcr analysis nm forward and reverse primers, μl of template dna and syber green reaction master mix (applied biosystems, grand island, ny, usa) were used in a final volume of μl. as previously described [ ] , we used the targets the dna polymerase gene l. chagasi (genbank accession: af ) and the pair of primers (forward: ' tgt cgc ttg cag acc aga tg '; reverse: ' gca tcg cag gtg tga gca c '). the pcr reactions employed an initial denaturation at °c ( min), denaturation cycles at °c ( seconds), in addition to annealing and extension at °c ( min.). the pgemh t plasmids (promega) was used for constructing stardart curves, for each run, containing inserts of interest [ ] . the gapdh gene ( -bp fragment genbank accession number ab ) was used to analyze the integrity of the samples. the primers 'ttccacggcacagtcaag ' (forward) and ' actcagcaccagcatcac ' (reverse) were used for gapdh gene amplification. the spleen parasite load was performed in duplicate and calculated by interpolation from the standard curve included in the same experimental batch. the data was expressed as number of l. chagasi organisms/ ng of total dna. the prism . software package (prism software, irvine, ca, usa) was used to evaluate data distribution normality by kolmogorov-smirnoff test and for further statistical analyses. oneway analysis of variance (anova) followed by tukey's multiple comparison were used for comparisons of cytokine profiles, no levels and parasite load amongst the experimental groups. student's t-test was used for intra-group comparisons between the control cultures (cc) and antigen-stimulated cultures (vsa or slca-stimuli in vitro). pearson correlation analysis was further applied to evaluate the relationship between spleen parasite burden and no profiles. additionally, the cytokine networks were assembled using cytoscape software version . . (institute of systems biology, seattle, usa), for each experimental group ("control", "sal", "lbsal", and lbsapsal) in all times analyzed (t , t rd , t , and t ), based on the correlations indices obtained by pearson correlation analysis. the network constructed using distinct edges between nodes to identify negative or positive correlations, referred as moderate ( . <r> . ) or strong (r> . ). in all cases, the significance were considered at p< . . "lbsapsal" immunization induced increased levels of ifn-γ before and after experimental l. chagasi-challenge aiming to verify whether the immunization protocols were able to induce the production of pro-inflammatory cytokines, we evaluated the profiles of tnf-α, ifn-γ, and il- in cc or upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t ) ( fig a, b and c, upper panel) . data analysis performed at (t rd ) post-vaccination did not show any differences in the tnf-α levels amongst the experimental groups, regardless the culture conditions. analysis of il- demonstrated that the "lbsapsal" group displayed increased levels (p< . ) upon vsastimulation as compared with the same cultures in the "control", "sal", and "lbsal" groups ( fig b, middle panel) . moreover, the "lbsal" group showed reduced il- levels (p< . ) upon slca-stimulation as compared with the "control" group ( fig b, middle panel) . interestingly, the analysis of ifn-γ showed that "lbsapsal" group presented increased levels (p< . ) in cc as compared to the "control" group ( fig c, middle panel) . additionally, "lbsapsal" group displayed increased ifn-γ levels (p< . ) upon vsa and slca-stimuli in comparison with "control", "sal", and "lbsal" groups ( fig c, middle panel) . analysis of the cytokine profile early after l. chagasi-challenge (t ) demonstrated that "lbsapsal" group showed a significant increase of tnf-α levels (p< . ) upon vsa-stimulation as compared to "sal" and "lbsal" groups ( fig a, middle panel) . no differences in the il- levels were observed amongst the experimental groups, regardless the culture conditions. analysis of ifn-γ, "lbsapsal" group displayed increased levels (p< . ) upon vsa-stimulation as compared to "control" and "lbsal" groups at t (fig c, middle panel) . additionally, "lbsapsal" group showed a significant increase of ifn-γ (p< . ) upon slca-stimulation as compared with the "control" group ( fig c, middle panel) . data analysis late after l. chagasi-challenge (t ) demonstrated that the "lbsal" group displayed increased tnf-α levels (p< . ) upon slca-stimulation as compared with the "control" group ( fig a, bottom panel) . in addition, the "lbsapsal" group displayed higher levels of tnf-α (p< . ) upon vsa-stimulation as compared with "lbsal" group ( fig a, bottom panel) . no differences in the il- levels were observed amongst the experimental groups, regardless the culture conditions. interestingly, the analysis of ifn-γ revealed that "lbsapsal" and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for tnf-α (a), il- (b) and ifn-γ (c). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" and "lbsal" groups, respectively. group showed higher levels (p< . ) upon vsa-stimulation as compared with the "control", "sal" and "lbsal" groups ( fig c, bottom panel) . "lbsapsal" induced lower levels of il- and tgf-β but while unvaccinated dogs presented higher amounts of il- after l. chagasichallenge aiming to evaluate whether the immunization protocols would induce regulatory/anti-inflammatory cytokines, we further quantified the levels of il- , il- , and tgf-β upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t ) (fig b, upper panel) . the results observed at the post-vaccination period (t rd ) demonstrated that the "lbsal" group showed a significant reduction in the il- levels (p< . ) upon vsa-stimulation as compared to the "sal" group ( fig b, middle panel) . no differences in il- and tgf-β levels were observed amongst the experimental groups, regardless the culture conditions. data mining performed early after l. chagasi-challenge (t ) revealed no differences in the il- production amongst the experimental groups, regardless the culture conditions. however, "lbsapsal" group showed a significant reduction of il- levels (p< . ) upon slca-stimulation as compared to the "sal" group (fig a, middle panel) . interestingly, "lbsapsal" group also presented a significant reduction of tgf-β levels (p< . ) upon slca-stimulation as compared the "control" group (fig , middle panel) . analysis carried out late after l. chagasi-challenge (t ) did not differences in il- levels amongst the experimental groups, regardless the culture conditions. the results showed that the "lbsal" group presented a significant reduction in the il- levels (p< . ) upon vsa-stimulation as compared with the "sal" group ( fig b, bottom panel) . interestingly, "lbsapsal" group displayed a significant reduction of tgf-β levels (p< . ) upon slca-stimulation as compared with the "control" group (fig , bottom panel) . the hallmark of the cytokine network in the "lbsapsal" group is a balanced immune response regarding positive correlation between proinflammatory cytokines (il- , ifn-γ, tnf-α) and regulatory cytokines (il- ) aiming to identify the overall balance of the evaluated cytokines, we have applied machinelearning approaches to analyze the cytokine network for all immunization protocols generated upon slca and vsa-stimuli in vitro (s fig and fig ) . the cytokine balance was calculated as the ifn-γ/il- and ifn-γ/il- ratio for all experimental groups and culture conditions and data provide in the s fig. the results re-enforce that ability of "lbsapsal" vaccine to induce a long lasting ifn-production over the synthesis of both il- and il- , regardless the antigen-stimuli (s fig). data mining by system biology tools were used to generate biomarker networks for each experimental group using both vsa and slca-stimuli. the results demonstrated that upon vsa-stimulation, the "control" group presented negative correlations between il- with tnfα and il- (vsa stimulus). positive correlation was also observed between tnf-α and ifn-γ. upon slca-stimulation, positive correlation was observed between il- and ifn-γ along with a negative correlation between tnf-α and il- (fig , upper left panel) . the "sal" group presented, upon vsa-stimulation, a range of positive correlations, including tnf-α with il- , ifn-γ and il- along il- with il- . in addition, negative correlation was observed between ifn-γ and il- . analysis upon slca-stimulation demonstrated data were analyzed at baseline before vaccination (t ), days after third immunization dose (t rd ) as well as early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge.the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for il- (a) and il- (b). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbol sal indicates significant differences in comparison to the "sal" group. positive correlations amongst the pro-inflammatory cytokines (tnf-α, ifn-γ and il- ) along positive correlation of them with il- (fig , upper right panel) . analysis of the "lbsal" group upon vsa-stimulation demonstrated a single negative correlation between tnf-α and il- (fig , bottom left panel) . "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the xaxis displays the different experimental groups ("control" and "lbsapsal") according to the in vitro stimuli (control culture [cc] and slca). the y-axis represents the tgf-β levels (pg/ml). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc or slca-stimulated cultures. the symbol c indicates significant differences in comparison to the "control" group. data analysis demonstrated that upon vsa-stimulation, the "lbsapsal" group showed strong positive correlation among tnf-α and ifn-γ along with positive correlation between tnf-α and il- . negative correlations were also observed amongst tnf-α and ifn-γ with il- (fig , bottom right panel) . moreover, upon slca-stimulation, positive correlation was observed between il- with ifn-γ and il- (fig , bottom right panel) . a sustained prominent reduction in spleen parasite load was observed in "lbsapsal" group later on after l. chagasi-challenge the parasitological analysis performed in spleen samples later on (t ) following l. chagasichallenge and reported as number of l. chagasi organisms/ ng of total dna in spleen samples and presented in table . data analysis demonstrated that all vaccination protocols were able the groups are represented as follows: "control"; "sal" (salivary glands of lutzomyia longipalpis); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands) and "lbsapsal" (antigen of l. braziliensis plus saponin and lutzomyia longipalpis salivary glands). the letter "a" indicate significant difference in relation to the sal, lbsal and lbsapsal groups. reduction (%) in parasite load was calculated as the proportion of number of amastigotes organisms/ ng of total dna observed in "sal", "lbsal" and "lbsapsal" groups in relation to "control" group. no clinical signs and mortality were observed throughout the experimental design. doi: . /journal.pone. .t to induce a reduction in the splenic parasite load as compared to the "control group". indeed, "sal" and "lbsal" groups yielded . % and . % of reduction in the spleen parasite load ( . and . amastigotes/ ng of total dna, respectively) as compared to the "control" group ( . amastigotes/ ng of total dna). "lbsapsal" groups showed the lowest parasite load ( . amastigotes/ ng of total dna), leading to . % of reduction rate in relation to the "control" group (table ) . moreover, no clinical signs and mortality were observed throughout the experimental design (table ) . "lbsapsal" group showed after l. chagasi-challenge enhanced no production with negative association with the spleen parasite load the levels of nitric oxide produced by pbmcs were evaluated upon vsa or slca-stimuli in vitro early (t ) and late (t ) after l. chagasi-challenge. data analysis carried early (t ) after l. chagasi-challenge, demonstrated that the "lbsap-sal" group presented upon control culture condition enhanced no levels (p< . ) as compared to the "sal" group ( fig , upper left panel) . additionally, "sal", and "lbsapsal" groups showed increase in the no levels (p< . ) upon vsa-stimulation as compared to the "control" and "lbsap" groups ( fig , upper left panel) . the analysis performed late (t ) after l. chagasi-challenge showed the "lbsapsal" group displayed higher no levels (p< . ) upon slca-stimulation as compared to the "control" group ( fig , upper right panel) . additional analysis revealed that the no production at t displayed a negative correlation with the spleen parasite load selectively upon slca-stimulation (fig , bottom right panel) . interestingly, it was observed that in the "lbsapsal" group, four out of five animals ( %) showed simultaneous high no production and low spleen parasite load, compatible with a protection profile (fig , bottom right panel) . in contrast, in the "control" group, only one out of five ( %) showed high no production but all animals presented high spleen parasitism (fig , bottom right panel) . the control of leishmania chagasi (syn. leishmania infantum) infection in dogs is essential to stop the current spread of zoonotic visceral leishmaniasis. therefore, a vaccine against vl would be an important tool for controlling cvl and would dramatically decrease anxiety regarding l. chagasi infection in humans [ , ] . in this sense, the establishment of biomarkers of immunogenicity is considered critical in the rational approach for analyzing candidate vaccines against cvl, and it contributes to identifying the pattern of immune response in dogs and the search for vaccine candidates against cvl [ , ] . for this reason, in this work, the immunogenicity and protective effects of the "lbsapsal" vaccination in dogs were investigated using levels of no and cytokines and evaluations related to the spleen parasite load. furthermore, the addition of sand fly saliva extract to vector-based vaccines can enhance the ability of the host to control or block leishmania infection [ , ] . the major results observed after "lbsapsal" immunization revealed a reduction in il- levels during the early (t ) post-challenge period. importantly, previous studies have identified that the presence of il- in splenocytes from dogs naturally infected with l. chagasi would be an important biomarker during ongoing cvl [ , ] . however, in some reports, the role of il- was related to the resistance profile or susceptibility profile of cvl [ , ] ; the higher levels of il- are considered the hallmark of dogs naturally infected with l. chagasi [ ] and sustained reduction of il- has been also reported by other immunogen candidates. in fact, reduction in the levels of il- after vaccine immunization against cvl has been considered a biomarker for protection against leishmania infection [ ] . in the present study, the evaluation of il- demonstrated a possible association with events related to the susceptibility to infection by l. chagasi; the "control" group has presented increased amounts during the late (t ) post-challenge period. in fact, this cytokine has been fig . impact of distinct immunization protocols on the no production elicited early and late after l. chagasi-challenge. no levels (μm) were determined in supernatants from pbmcs cultures maintained upon vaccine-soluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). top panels: the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the nitrite levels [μm] . data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" or "lbsal" groups, respectively. bottom panels: correlation between no levels and spleen parasite load (# amastigotes/ ng of total dna) at t considering cc (bottom left panel) or the presence of a stimulus (vsa: bottom middle panel; or slca: bottom right panel) in all groups. the groups are distinguishable by colors as follows: as follows: "c" (white circles); "sal" (ligh gray circles); "lbsal" (dark gray circles) and "lbsapsal" (black circles). the quadrants represented in the bottom panels delimit the low and high no producers (y-axis) and the low and high spleen parasite load (x axis). associated with severity of vl [ ] and cvl [ , , , , , ] . importantly, the "lbsapsal" group did not have increased il- production, even after the late l. chagasi-challenge period. additionally, analysis of tgf-β revealed reduced production in the "lbsapsal"-immunized group during the early and late post-challenge periods. interestingly, the presence of tgf-β has been associated with inducing immunosuppression characteristics during the course of vl [ , ] . moreover, the presence of tgf-β in vitro has a protective effect for amastigotes in macrophages, favoring the maintenance of parasitism [ ] . in addition, alves et al. [ ] reported high levels of tgf-β associated with increased parasite load in lymph nodes from symptomatic dogs and concluded that tgf-β is associated with morbidity in cvl. in this context, it is possible to hypothesize that lower levels of tgf-β in the "lbsapsal" group would indicate the establishment of immunoprotective mechanisms, whereas higher amounts in the "control" group would be associated with the susceptibility pattern after l. chagasi-challenge. analysis of pro-inflammatory cytokines has been considered a prerequisite for composing immunogenicity analyses before and after experimental challenge with l. chagasi in clinical trials of anti-cvl vaccines [ , ] . the analysis of tnf-α before vaccine immunization (t ) demonstrated increased levels in slca-stimulated cultures in the "control" and "lbsapsal" groups and demonstrated a tendency for enhanced amounts in the "sal" and "lbsal" groups. similar results were observed for il- and ifn-γ at t . these results seem to indicate that slca stimulation would induce increased levels of these cytokines, pointing to an inherent feature of the antigenic stimulus. for this reason, we evaluated all groups and analyzed the same stimulus in each group to identify a cytokine profile regarding type i immune response. this strategy demonstrated enhanced levels of il- and ifn-γ post-vaccination and sustained production of both tnf-α and ifn-γ even after early and late post-challenge periods in the "lbsapsal" group. some studies have described tnf-α as being associated with a resistance profile in cvl [ , , , , ] or in dogs vaccinated against cvl [ ] , and has been associated with susceptibility when associated with high levels of il- and il- [ ] . results obtained by strauss-ayali et al. [ ] showed that after stimulation with exogenous il- , pbmcs from l. infantum−infected dogs were able to reverse an apparent state of anergy, resulting in increased production of ifn-γ. moreover, menezes-souza et al. [ ] found that low levels of il- concomitant with high levels of il- and tgf-β represent a favorable condition for the persistence and replication of parasites in cvl. it has been described that ifn-γ is linked to a resistance profile in different experimental models for vl [ , , , , ] and in dogs vaccinated against cvl [ , , , , ] . our data regarding the cytokine network indicated a balanced immune response in the "lbsapsal" group. in this sense, we describe positive (tnf-α versus ifn-γ and il- ; il- versus ifn-γ and il- ) and negative (il- versus tnf-α or ifn-γ) correlations demonstrating a prominent pro-inflammatory immune response in the "lbsapsal" group. in addition to a non-significant increase in il- or il- levels in the "lbsapsal" group, a balanced immune response was demonstrated by taking into account the positive correlations between il- and type i cytokines (ifn-γ, il- , tnf-α). these data should be related to the regulation of the prominent pro-inflammatory immune response induced by the "lbsapsal" vaccination and should aim to control potential tissue damage by type i cytokines. the parasitological evaluation revealed in "lbsapsal" group a remarkable reduction in spleen parasite load ( . %) after experimental l. chagasi-challenge, in concordance with a prominent pro-inflammatory immune response induced by this vaccine. in fact, we have been described a parasite load reduction of % in the lbsapsal group [ ] , indicating the capacity of this vaccine to control parasite replication even long after challenge ( days). sand fly saliva displays an important role in the first steps of leishmania infection, due to the vast repertoire of pharmacologically active molecules that surround host's hemostatic system [ ] . the re-exposure to the salivary components seems to display an immunogenic activity, eliciting antibody production and cell mediated immunity by the host that could be block or limit the leishmania infection [ ] . some studies have been shown that l. longipalpis salivary proteins induce an immune response associated with protection in dogs [ , ] . furthermore, in a hamster model, salivary proteins of a sand fly protects against the fatal outcome of visceral leishmaniasis [ ] . similarly, we observed . % parasite load reduction in "sal" group, showing that salivary components have a high potential to limit infection in dogs. the experimental challenge in vaccine studies against canine visceral leishmaniasis is considered as crucial to analyze the protection performance. distinct studies have been published aiming to determine the experimental l. chagasi-challenge plus sand fly saliva using intradermal route in dogs would be more similar to natural infection than intravenous challenge [ , ] . however, using intradermal challenge, the dogs would be asymptomatic during all the study, besides to present lower parasitism [ ] . since the "lbsapsal" vaccine presents saliva as antigenic compound, the ideal experimental challenge to test the protection should ideally be performed by intradermal route, as analyzed in our study. importantly, we observed increased no levels in the "lbsapsal" group during the early and late post-challenge periods. interestingly, four out of five dogs immunized with "lbsapsal" presented higher no amounts and low spleen parasite burden during the late post-challenge period, indicating long-term immunogenicity and resulting in reduction of parasitism. in fact, we have previously demonstrated that "lbsapsal" induced resistance biomarkers specifically related to expansion of circulating cd + and cd + t-cells and leishmania-specific subsets and lower levels of parasitism [ , ] . panaro et al. [ ] also observed an increase in no production and anti-leishmanial activity of macrophages, as well as increased levels of ifn-γ in pbmcs supernatants, in dogs immunized with a vaccine comprising crude antigens of l. infantum. taken together, our major data indicate that immunization with "lbsapsal" is able to induce a protection profile characterized by enhanced amounts of type i (tnf-α, il- , ifnγ) cytokines and reduction in type ii cytokines (il- and tgf-β), even after experimental challenge. the establishment of a polarized type i immune response after "lbsapsal" immunization supported increased levels of no production, favoring a reduction in parasitism and indicating long-lasting protection against l. chagasi infection. these results encourage further studies that can provide important information for a better understanding of the effectiveness of the "lbsapsal" vaccine and strategies for addressing leishmania antigens in combination with sand fly proteins such as those present in the saliva in the vector. supporting information s fig. impact of distinct immunization protocols on the pro-inflammatory/regulatory cytokine balance. the balance of inflammatory cytokine ifn-γ and regulatory/anti-inflammatory (il- and il- ) were analyzed in the supernatant of pbmcs maintained upon vaccinesoluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine ratio (ifn-γ/il and ifn-γ/il- ). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) amongst the cc, vsa or slca-stimulated cultures. the symbols c and sal indicate significant differences in comparison to the "control" or "sal" groups, respectively. 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high antibody production in balb/c mice caused by natural exposure to lutzomyia longipalpis bites a follow-up of beagle dogs intradermally infected with leishmania chagasi in the presence or absence of sand fly saliva experimental infection of dogs with leishmania and saliva as a model to study canine visceral leishmaniasis nitric oxide production by macrophages of dogs vaccinated with killed leishmania infantum promastigotes the authors are grateful for the use of the facilities at cebio, universidade federal de minas gerais, rede mineira de bioterismo (fapemig) and universidade federal de ouro preto. this work was supported by fundação de amparo a pesquisa do estado de minas gerais, bra- key: cord- - x f t authors: lin, feng; muthuraman, kumar; lawley, mark title: an optimal control theory approach to non-pharmaceutical interventions date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: x f t background: non-pharmaceutical interventions (npi) are the first line of defense against pandemic influenza. these interventions dampen virus spread by reducing contact between infected and susceptible persons. because they curtail essential societal activities, they must be applied judiciously. optimal control theory is an approach for modeling and balancing competing objectives such as epidemic spread and npi cost. methods: we apply optimal control on an epidemiologic compartmental model to develop triggers for npi implementation. the objective is to minimize expected person-days lost from influenza related deaths and npi implementations for the model. we perform a multivariate sensitivity analysis based on latin hypercube sampling to study the effects of input parameters on the optimal control policy. additional studies investigated the effects of departures from the modeling assumptions, including exponential terminal time and linear npi implementation cost. results: an optimal policy is derived for the control model using a linear npi implementation cost. linear cost leads to a "bang-bang" policy in which npis are applied at maximum strength when certain state criteria are met. multivariate sensitivity analyses are presented which indicate that npi cost, death rate, and recovery rate are influential in determining the policy structure. further death rate, basic reproductive number and recovery rate are the most influential in determining the expected cumulative death. when applying the npi policy, the cumulative deaths under exponential and gamma terminal times are close, which implies that the outcome of applying the "bang-bang" policy is insensitive to the exponential assumption. quadratic cost leads to a multi-level policy in which npis are applied at varying strength levels, again based on certain state criteria. results indicate that linear cost leads to more costly implementation resulting in fewer deaths. conclusions: the application of optimal control theory can provide valuable insight to developing effective control strategies for pandemic. our findings highlight the importance of establishing a sensitive and timely surveillance system for pandemic preparedness. emerging influenza is threatening the world with the next pandemic [ ] . the current swine flu caused by a novel h n virus has infected a documented , humans, killing , from april to august [ ] . the world health organization (who) declared the outbreak to be a pandemic because of growing worldwide cases [ ] . currently, the severity of the outbreak is moderate as most people recover from infection without the need for medical care [ ] . however, if the virus mutates and achieves the ability to cause severe illness, it will kill more people and overwhelm the health system. vaccination is the most effective means of pandemic mitigation. vaccine production is a complex multi-step process which involves development, manufacturing, and delivery processes and current levels of vaccine production capacity are inadequate. thus many uncertainties exist in every step and effective vaccines are typically available well after the viral strain has emerged [ ] [ ] [ ] [ ] . for instance, vaccines against the h n strain are still under development and will remain in short supply by november [ ] . current stockpiling of antiviral drugs will also be in short supply and their efficiency will be limited once a pandemic occurs [ , ] . public health systems need to be prepared for cases when effective pharmaceutical interventions are unavailable. non-pharmaceutical interventions (npis) are necessary to delay and dampen the pandemic before pharmaceuticals become available [ ] . recommended npis include: ( ) social distancing: school closure, workplace distancing, restricted public gathering and travel; ( ) case containment measures: voluntary case isolation, voluntary quarantine of members of households with ill persons; and ( ) infection control measures: hand hygiene, cough etiquette, and mask/respirator usage [ , ] . npis were implemented during the pandemic and more recently during the severe acute respiratory syndrome (sars) outbreak of . although research on these events confirms the importance of npis, suboptimal triggering during the pandemic rendered npis only moderately effective at reducing mortality [ ] [ ] [ ] . during sars, sheltering and quarantine were found to be effective [ , ] , while border screening was not [ ] . during the current h n outbreak, infection control is recommended to prevent spread of the virus among humans. public health authorities are developing action plans which may request social distancing actions depending on the severity of the outbreak [ ] . mathematical models are often used to study disease spread, with the susceptible-infectious-recovered (sir) model being preferred for diseases spread via droplet and aerosol. the sir model has been used to study pandemic flu [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , seasonal flu [ ] [ ] [ ] , sars [ , [ ] [ ] [ ] , and smallpox [ ] [ ] [ ] [ ] . these papers use sir to simulate the disease outbreak and evaluate the effectiveness of selected control measures under various predefined scenarios. they do not provide optimal controls for initiating implementation, and thus we will not review them here. sir literature most directly relevant to this work includes [ ] [ ] [ ] [ ] [ ] [ ] . these authors use the sir model to study optimal controls, i.e., controls that minimize a prescribed objective function. most show "bangbang" controllers to be optimal (in the sense of minimizing a specified objective function). these policies apply no control until the occurrence of a triggering event and then apply controls at maximum strength. sethi derived optimal closed-form results for isolation and immunization policies [ , ] using an si model. with this model, the population is partitioned into two parts, susceptible and infectious. the control is to either isolate and vaccinate at a maximum rate or do nothing. infectious individuals who recover become susceptible once again, and thus immunity due to infection and subsequent recovery are not considered. clancy [ ] studied the properties of optimal policies for isolation and immunization assuming that all infectious individuals can be immediately isolated and all susceptible individuals can be immediately immunized. the policy takes no action when the number of infectious is below an optimal threshold and immediately isolates and/or immunizes when the number exceeds the threshold. however, they can only obtain optimal policies when the state space is small. morton and wickwire [ ] developed optimal control policies for immunization assuming an infinite pandemic terminal time. however their switching curve derivation has an error in the derivatives (eqs. a and b of [ ] ) and thus their results are unclear. behncke [ ] derived mathematical properties of optimal vaccination programs under the following assumptions: ) the time when vaccine becomes available is known; ) infectious individuals can be immediately and completely isolated; and ) the time horizon of the pandemic is infinite. overall, we think the underlying assumptions of currently published results are questionable. it is not the case that all infectious can be immediately identified and isolated. further, planners do not know when vaccines will become available, and even when available, it is not true that mass prophylaxis is instantaneous. finally, during a pandemic, people will die. the current models do not account for mortality, which could be significant for viral strains such as h n . in this work, we use an expanded sir model to develop triggers for npi implementation to minimize expected person-days lost resulting from influenza related deaths and npi implementation. npi policies are derived for a deterministic control model. results are compared with the most relevant optimal control papers discussed above. multivariate sensitivity analyses based on latin hypercube sampling are performed to investigate the effects of input parameters on the control policy structure and the mean cumulative deaths. additional studies investigate the effects of departures from the modeling assumptions, which include exponential terminal time and linear npi implementation cost. in this section, we formulate an optimal control problem with an expanded epidemic model to compute npi implementation strategy. to understand the following discussion, the reader is referred to figure , which illustrates the compartmental model, and to table , which provides a summary of notation. to construct the model, we make six assumptions: . at any time, t ≥ , the community is composed of s(t) susceptible, i(t) infectious, r(t) recovered, and d(t) deceased individuals. the population is closed, ignoring the demographic turnover or immigration, i.e. s(t) + i(t) + r(t) + d(t) = n. to make the analysis independent of population size, we normalize the model by letting s t . thus, any community can be described by state variable x(t) = (s(t), i(t), r(t), d(t)). . the population is homogeneously mixed and people make contact at random. . people susceptible are able to get infected when they contact infectious people. once infected, they move into the infectious compartment. people infected can either recover at a constant rate, g, or die at a constant rate, τ. people recovered are assumed to be immune within our study horizon. . npi implementation is modeled by the decision variable u(t), where ≤ u(t) ≤ b <b. npi implementation reduces the rate of contact between susceptible and infectious individuals, and thus dampens the infection rate from b to a lower level, bu(t). in this paper, we only consider npis that will disrupt the normal societal functions and result in significant economic impact, such as school closure, work space distancing and voluntary isolation and quarantine. we assume these npis have the same effect in reducing disease spread. . we let person-days lost due to death be unit cost, while c ≤ will be person-days lost due to npi implementation. the cost c is a weight of npi cost relative to death. it measures the loss of productivity (persondays) due to implementing npis. to determine the value of c, the public health officials need to consider many factors, such as culture of the community, perceptions to death, consequences of pandemic and of figure expands the classic susceptible-infectious-recovered (sir) model to capture the mortality. table model notation. provides a summary of notation. x(t) = (s (t), i(t), r(t), d(t)) state that describes the disease status of a community x( ) = (s ( ), i( ), r( ), d( )) initial disease state of a community proportion of the control space hamilton-jacobi-bellman equation the npis, economic constraints, and etc. we assume that cost of npi implementation increases with implementation strength. in particular, we consider two cost structures, a linear structure in which cost is proportional to npi strength, and a quadratic structure in which the change in cost with npi strength is more variable. this paper is focused most extensively on the linear structure. . the optimization horizon t is assumed to be the time when effective vaccine becomes available to the community. although the production of the first doses of vaccine is estimated to take - months once the virus strain can be identified, nobody knows exactly when circulating virus can be identified. along with vaccine development, there are issues regarding manufacturing, delivery, and deployment. to capture the uncertainty in vaccine arrival time, we let t follow an exponential distribution. we can derive a model with a more general vaccine arrival time, but the problem is not currently numerically tractable, that is, algorithms for the general terminal time case present significant challenges and yet to be developed and tested. the exponential assumption ensures we get the discounted value function, which allows for a solution and some insights about the policy. in later sections we tested the model sensitivity to the exponential assumption. the probability density function (pdf) of t is written as f(t, Φ) = Φe -Φt , where t ≥ . because npi implementation disrupts daily activities vital to a productive society, they should only be used when they will be effective. thus, in developing triggers, npi cost must be balanced with npi effectiveness. in this work, we use person-days lost to measure npi cost and effectiveness. npis cause person-day losses by shutting down vital activities, but mitigate person-day losses by reducing mortality. by capturing the dynamic relationship between npi implementation, mortality, and associated person-day losses, our model provides a mechanism for finding an npi triggering scheme that helps minimize total person-days lost during the time period before vaccines become available. the dynamics of the controlled epidemic are given by the non-linear differential equations (dots denote time the total person-days lost for a pandemic under a policy which prescribes an admissible control, u, will be denoted by the value function v (x, u), which is defined as: the objective is to minimize expected person-days lost over the time horizon, t, which is assumed to be the time when effective vaccine becomes available to the community. an admissible control, u*, which minimizes v (x, u) will be called optimal, and the corresponding value function is denoted by: from eqs. ( ) and ( ) we can see that the knowledge of the state variables s(t) and i(t) determines the other two, thus we focus our analysis on the susceptible and infectious compartments. this reduces the system to two dimensions, s and i. thus, we study the control system in the (s, i)-plane, defined as Ω = {(s, i), s, i ≥ , s + i ≤ }. we let (s, i) represent the initial state x( ) = (s( ), i( )), and v (x, u) = v ((s, i); u) be the associated value function, which captures total person-days lost starting in state (s, i) Ω and operating under control u. the expected value of v*(s, i) is calculated by integrating v ((s, i), u)·f(t; Φ) over the range of t: by changing the order of integration, the problem is converted to an infinite horizon discounted problem: based on pontryagin's maximum principle [ ] , which gives necessary conditions for optimal control, we can derive a first-order partial differential equation satisfied by the optimal value function, v*. this is called the hamilton-jacobi-bellman (hjb) equation: eq. ( ) is a linear function of u, thus the optimal control will be "bang-bang" control [ ] , i.e., the candidate control, u*, should satisfy: where the switching curve ψ (s, i) is the coefficient of u in eq. ( ), defined as: the optimal control u* is pushed to its lower/upper bound depending on the sign of its coefficient, the switching curve ψ (s, i), because the hjb equation, eq. ( ), is linear in the control, u. however, if the hjb is nonlinear in u, the optimal control will not be bangbang. an example will be the linear quadratic optimal control problem, where the optimal policy is a linear state feedback [ ] . by the bang-bang nature of candidate u*, the npis should be implemented at the maximum level when ψ (x) < . to understand this, assume the community is in state x a = (s, i) and consider exerting an instantaneous and small control, δ u , which will move the system to state x b = (s + δ u si, i-δ u si) instantaneously. the exertion will cost csδ u person-days lost. if v*(x a )-v* (x b ) exceeds csδ u , then it is cost-effective to implement the npis. hence, indicates that exerting control reduces person-days lost. we keep implementing npis until ψ (x) > . due to the complexity of the eq. ( ), the switching curve, ψ (x), has no closed form. to find ψ (x), we use an algorithm based on dynamic programming [ ] , which requires discretization over time and space. the uniqueness and convergence of the solution is guaranteed by the viscosity solution concept developed in [ ] . the optimal control, u*, is obtained by solving the hjb equation (eq. ( )). figure shows u* for two infection rates, . and . , given a recovery rate of . and a death rate of . (taken from [ ] ). person-days lost from npi implementation, c, is set to . ( % of the cost of a single death). the maximum impact of npis on the infection rate is assumed to be a % reduction. note that the basic reproductive number r without any control is given by figures (a) and (b) indicate when to trigger npi implementation. when the system state falls in region Ω , npis should be implemented at maximum strength; in contrast, npis should not be implemented when the state falls in Ω . for example, in the influenza scenario of figure (a), npis should be implemented when % remains susceptible and % of the population is infected. however, if % remains susceptible and % is infected, it is better not to trigger the npis. we compare our policy against the most relevant optimal isolation policies derived in [ ] . figures (c) and (d) show these isolation policies under the different infection rates. the control either isolates at a maximum rate when the number of infectious exceeds a threshold or does nothing. for a pandemic with b = . and r = . , figure (d) tells us not to act until the percent infectious exceeds %; while figure (b) tells us to implement npis at an earlier stage of the outbreak, for example % susceptible and % infectious. we illustrate cases for which r > , i.e., the uncontrolled infection spreads rather than dying out. figures (a) and (b) compare the epidemic curves with and without npis, starting from a state % susceptible and % infected. according to figures (a) and (b) , the npis should be triggered at this state. in figure (a) , npi implementation not only reduces the total death by %, but also eliminates the peak of the outbreak. overall, npi implementation saves % of the average person-days lost. in figure (b) , where a more severe pandemic is considered, the reduction in total deaths is % and npi implementation reduces and delays the peak of outbreak, which allows additional time for vaccine development. figures (c) and (d) compare the epidemic curves with and without npis starting from states that fall on the control thresholds of [ ] shown in figures (a) and (d) . the proportions of recovered and dead population were set to because they could not be differentiated from infectious people in an si model. it is still best to implement npis at these states but the impact is limited. we also computed control policies for systems assuming quadratic control cost instead of linear cost, i.e., the value function is written as , while the system dynamics still follow eq. ( ). based on pontryagin's maximum principle [ ] , we can derive the hamilton-jacobi-bellman (hjb) equation for the new problem: eq. ( ) is now a quadratic function of u, thus the optimal control will not be "bang-bang" control. the candidate control u* should satisfy: by solving eq. ( ), we obtained the npi policies for systems assuming quadratic control cost shown in figure shows the optimal control policies for two infection rates, . and . , given a recovery rate g = . and a death rate τ = . . (a) presents the optimal npi control for b = . and r = . . (b) presents the optimal npi control for b = . and r = . . (c) presents the optimal isolation policy derived in [ ] for b = . and r = . . (d) presents the optimal isolation policy derived in [ ] for b = . and r = . . influenza characterized by b = . and r = . . figure (a) shows a multi-level policy in which npis are applied at varying strength levels based on certain state criteria. note that "red" indicates > % npi strength, "yellow" indicates % npi strength, and so forth. the level of npi policy decreases from % of maximum npi level to % as system state traverses from the upper to the lower part and from the right corner to the interior region. the contour line at u* ≈ % of maximum impelmentation level is very similar in shape and location to the boundary between Ω and Ω in figure (a). as another demonstration, figures (b) and (b) show the control policies under both control costs with influenza characterized by b = . . the two boundaries between Ω and Ω in figure (b) resemble the contour lines u* ≈ % of maximum impelmentation level in figure (b). the two regions of high level of u* in the upper corner and lower right corner of figure (b) correspond to two control regions Ω in figure (b). in addition, table compares the means of the expected person-days lost per person due to death, d , and control intensity, u *, between the linear and quadratic models. the overall npi strength of the linear model is higher than that of the quadratic model, while the expected person-days lost due to death is lower. the linear model tends to implement npis more intensely and save more lives while having a higher overall cost. a multivariate uncertainty and sensitivity analysis was performed to study the effects of input parameters on the control policy for the linear cost model. this analysis investigated the effects of five inputs (r , g, τ, c, b) on a performance measure, ω, defined as the proportion of the control space to the total state space, i.e., . there is no well-defined performance measure to evaluate the npi policy, especially when the policy is defined in a -dimensional state space. we figure epidemic curves of infectious and dead population with and without npi implementation. figure shows the impact of optimal control on pandemic severity, peak, and total deaths, when npis are triggered at different initial states. (a) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (b) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (c) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (d) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . chose ω because it captures the overall intensiveness of npi implementations. in addition, we investigated the effect of these parameters on the outcome of applying the control policy, defined as the mean cumulative death, d t . we simulated the sird system under the optimal policy starting from all state (s , i , r , d ), where s > %, i < %, and d = . the simulation was terminated at a randomly selected exponential terminal time, and we recorded and analyzed the cumulative number of deaths. the mean cumulative death was calculated by taking the average of cumulative deaths over all tested initial states. table summarizes the estimated probability distribution functions (pdfs) of five input parameters, assuming the input parameters are statistically independent. the pdfs of influenza transmission characteristics (r , /g, and /τ) are estimated based on the pandemic [ ] . note that the infection rate b can be written as r (g + τ). the effect of npis, b, was found to reduce the infection rate, b, by up to - % in and in many cases the effect of npis was very limited [ ] ; thus we assume the impact of npi implementation, b, follows uniform( , %). we are not able to find any empirical data on the cost of npi, c. this value is a relative cost, which depends on decision makers' perceptions of saving lives versus maintenance of daily societal functions. in our analysis we let c take the value from to . uniformly, which would imply that the cost of four sessions of maximal npi implementation is equivalent to the cost of one death. we sampled ranges of the parameters times using latin hypercube sampling (lhs) to generate scenarios [ ] [ ] [ ] . then we conducted multivariate uncertainty and sensitivity analysis to determine the uncertainty in the performance measure that was due to the uncertainty in estimating the input parameters. the descriptive statistics for ω and d t are given in table , which lists the mean, variance, median, minimum, and maximum of ω and d t . figure shows the empirical cumulative distribution functions (cdfs) of ω and d t obtained from lhs scenarios and table provides the partial rank correlation coefficients (prccs) for the performance measures and each parameter. the cdf of ω revealed a wide range of estimates due to the uncertainty in estimating the values of the five input parameters. sixty percent of ω estimates are less than . %, with a minimum of % and a maximum of . %. for ω, the prccs are all statistically significant, i.e. p < . . the cost of npi implementation, c, the time when a death occurs after infection, /τ, and the time for an infectious person to recover, /g, are the most statistically influential (|prcc| > . ). an increase in c or /τ corresponds to a decrease in ω; while an increase in the infection period /g corresponds to an increase in ω. the cdf of d t shows that the mean of cumulative death is . %, with a minimum of . % and a maximum of . %. for d t , the prccs are are all statistically significant, i.e. p < . . the most statistically influential inputs are /τ, r , and /g (|prcc| > . ) while the other two parameters are lessinfluential. a decrease in /τ corresponds to an increase in d t ; while an increase in r or /g corresponds to an increase in d t . to test the sensitivity of our control policy to the terminal time assumption, we simulated the disease propagation and studied the outcome of applying our npi policy to settings with an exponential and a gamma terminal time. for each flu scenario specified in the sensitivity analysis, a corresponding npi policy can be obtained. we randomly selected a value from exponential( . ) and a value gamma( , . ) as the vaccine arrival time (or simulation terminal time). the sampling was repeated times for each scenario. then, we simulated the sird system starting from an initial state applying the corresponding npi policy. the simulation was terminated at the sampled vaccine arrival times and the cumulative deaths were recorded. a total of initial states were selected, where s ≥ % and i ≤ %, for a total of , simulations. we studied the difference in cumulative deaths under these two vaccine arrival assumptions. table lists the descriptive statistics of percentage difference in cumulative deaths for the same initial states at two terminal times. overall, the difference in cumulative deaths under exponential and gamma terminal times is small (mean = . %). the distribution of difference in cumulative deaths is left-skewed, with . % of these differences being less than %. there are a few cases where the cumulative deaths differ significantly (≥ %). these cases all started from initial states where only a small table lists the partial rank correlation coefficients (prccs) for the performance measure ω and d t . table lists the summary statistics of difference in cumulative deaths at exponential and gamma terminal time. proportion people are infectious, i.e., i ≤ %, and the difference between the selected gamma and exponential terminal time exceeds % of the maximum of these two. for example, in a case where i = %, s = %, gamma terminal time = days and exponential terminal time = days, the difference in cumulative death is . %. effect of npi policies on the epidemic npis reduce and delay the spread of pandemic by moderating social contact between susceptible and infectious people. because npis disrupt daily societal functions, it is important that they be implemented judiciously. this requires identifying effective initiating triggers, which is a challenging research task. implementing npis will impede influenza spread; on the other hand, normal societal functions will be interrupted. the optimal control method takes both aspects into account and tries to find the best balance between them, given the decision makers relative weighting of the two. based on figures and , early implementation for moderate and severe pandemic is very important for npis to have impact on the outbreak and the impact is effective only if npis are implemented early. late npi implementation might still be optimal, but the impact is much less. for a severe pandemic, it is optimal to trigger npis at the beginning stage when susceptible population is large and infectious population is small. if we miss the beginning stage, it is not optimal to implement npis until the outbreak is significantly progressed. this is because once the pathogen achieves a certain level of infection, npis are not effective against it, and thus are not worth the cost. that is, the benefit of npis at a stage when the disease has progressed significantly is less than the cost of npi implementation. this finding supports the cdc pandemic mitigation guidelines, which state that when the pandemic is category or , all npis are recommended for early implementation [ ] . furthermore, earlier npi implementation reduces and delays the peak of the outbreak as illustrated figures (a) and (b), which allows additional time for vaccine development. if a severe pandemic occurs, hospitals will experience an overwhelming influx of patients and need to operate at their surge capacities. earlier npi implementation can reduce the magnitude of infectious at the peak, which relieves some of the burden on hospitals and other health care infrastructures. in contrast, npis are not nearly as effective if disease has already spread into the community as the cases shown in figures (c) and (d). in both cases, npis are triggered after the peak of the outbreak, where hospitals might have already been operating at their surge capacities for a few weeks. both cases start at states falling on the control thresholds recommended in figures (c) and (d) [ ] . this finding indicates that the additional complexity of our model is warranted when compared with the si model used in [ ] . timely and sensitive surveillance systems are key to successful application of the optimal control method as knowledge of both the pathogen characteristics and the community state are assumed. the surveillance systems should be able to identify the virus quickly and provide accurate estimates for parameters which characterize the severity of an influenza. the effectiveness of the control policy depends on the accuracy of these estimates, which include infection rate b, death rate τ and recovery rate g. once the control policy is computed, we also need to track the community state to determine if npis should be triggered. as early npi implementation is found to be much more effective, we do not want to miss the beginning stage of the outbreak. thus, the surveillance system should also estimate the community state, including the size of the infectious and susceptible populations. our sensitivity analysis identified three important input parameters for determining the overall npi intensity. the important parameters are the npi cost, death rate, and recovery rate. for high npi costs, it is not worthwhile implementing npis because the benefit is less than the cost. for higher death rates, the policy sacrifices daily societal functions to save lives. in contrast, when recovery rate is small, infected people recover more slowly and continue infecting susceptible people. thus, more npi implementation is required. these results suggest that an influenza virus with a high death rate and a small recovery rate requires early intensive npi implementation, particularly when the community places a high value on avoiding death. cumulative death was most affected by the death rate, the basic recovery number and the recovery rate. for higher death rates, a higher proportion of infected people will die. for higher basic reproductive number, more people will be infected, resulting in more deaths even when the death rate is smaller. for lower recovery rate, infected people recover at a slower rate, and thus more people will be infected. this suggests that an influenza virus with a high death rate, a high basic reproductive number and a small recovery rate is less affected by npi implementation. npi cost does not seem to affect the cumulative death. however, npi cost was identified as the most influential (prcc = - . ) in determining the intensity of npi implementation. different communities have different perspectives of death versus disruption of daily societal functions. the range of c should be determined by decision makers after carefully evaluating the demographic, cultural, and economic characteristics of the community. as a performance measure, ω does not capture the complete structure of the control policy. it only captures the overall control intensity, but subtle differences between control policies, such as distribution and shape of Ω in the state space, is missing. policies that have different structure in the state space might have same ω value. therefore, continued effort should be made in selecting more refined measures for the control policy performance. linear v.s. quadratic cost function if the cost function is linear, the control policy is bangbang, which suggests implementing npis at the maximum strength or not implementing at all as shown in figure . if the cost function is nonlinear, for example the quadratic cases presented in figure , the control policy has multiple levels, which requires varying the npi strengths as the system evolves from one state to another. it is easily shown from eqs. and that if the linear model indicates npi implementation for a state x, i.e., u* = b for state x, then the quadratic model also indicates some implementation in state x, i.e., u* > for state x. but the inverse is not true. this property can be proved easily. if u* = b in the linear model, we have ψ(s, i) < , for all (s, i) Ω according to eq. . so together with eq. , we have u* > for these states in the quadratic model. thus, a quadratic cost structure will require npis to be implemented in more states but with much lower intensity in those states. overall, a linear cost structure leads to higher average npi intensity than the quadratic cost. in the examples observed, the boundaries between control and non-control regions in linear model resemble the quadratic contour lines u* ≈ % of maximum implementation. moreover, the linear cost model appears to place more weight on death, thus implementing more control and saving more lives as shown in table . in cases with quadratic cost structure, using a linear model might result in over-control which would not be marginally effective. however, quadratic cost introduces additional complexity in parameter estimation, computation, and policy interpretation. for example, we must determine how to implement npis at x% of maximum. this requires determining which npis will be implemented for each control level. the bang-bang policy, on the other hand, has only two levels, which is easier to understand and implement. finally, it is not clear that a direct comparison between policies obtained under linear and qudratic cost structures is appropriate, because the value functions are defined differently. more research needs to be done to better define the npi levels and interpret the policy if a non-linear cost function is chosen. we are currently not able to derive an optimal control policy for a general terminal time assumption. although the exponential optimal policy will not be optimal for cases with general terminal time, results shown in table indicate that the expected cumulative deaths predicted by the exponential optimal policy will be close to those occurring in a more general terminal time case. this suggests that the impact of the optimal exponential policy on virus spread is not always sensitive to terminal time distribution. although the exponential assumption might not be completely realistic, making this assumption allows us to obtain the npi policy, which then seems to provide desirable impact for cases with nonexponential terminal times. our model was limited in several ways. first, although hjb can be derived for models assuming general terminal time (e.g., gamma), so far the control policy can only be computed assuming exponential terminal time. second, the present modeling framework does not capture uncertainty in parameter estimation, i.e. the model accuracy relies on accurate estimation of input parameters. in practice, collection of accurate data and estimation of input parameters from data can be challenging and time consuming. third, the present modeling framework assumes equal effect of various npis in a homogeneously-mixed population, while different npis will have distinct impacts for disparate population groups. finally, bang-bang control must be further refined since it is not clear that on/off implementation is realistic for larger communities. to better apply optimal control methods in disease control problems, continued efforts should be made to refine the present model and to better estimate the input parameters. to conclude, we have considered a problem of nonpharmaceutical intervention (npi) implementation for pandemic control using optimal control theory to develop triggers that minimize expected person-days lost associated with infection related death and npi implementation over an exponential time horizon. the best control strategy for the model depends on the transmission characteristics of the influenza virus, the state of the pandemic, and the cost and implementation levels of npis. we present the computed policies under different transmission characteristics, where it is optimal to activate all npis when the system state falls in the control region, Ω . the optimal policy can be calculated for any combination of flu and cost parameters. we compare the impacts of npis triggered at different states, which supports the idea of early containment. for comparison, we present the npi policies assuming quadratic control cost. the quadratic cost assumption introduces additional complexity into parameter estimation, computation and policy interpretation, thus more research needs to be done to better define the npi levels and interpret the policy. we perform multivariate sensitivity analysis, which identifies important parameters that affect the intensity of control and the outcome of applying the policy. the findings highlight the importance of establishing a sensitive and timely surveillance system. finally, we study the outcome of applying our npi policy under exponential and gamma terminal times, and find small difference in the cumulative death. many uncertainties exist in estimating flu parameters, future research directions include developing a model that allows using stochastic rather than deterministic inputs and updates the control polices in real time. since npi implementation is not mandatory, compliance to npi requirements is crucial for successful implementation. community engagement, job security, and disruption of daily life affect compliance to npi implementation [ ] . moreover, prolonged outbreak might result in compliance fatigue. thus, in future work, we will integrate time-based compliance models into the system dynamics. other important research directions include consideration of population heterogeneity, stochasticity and partial observability in disease outbreak, and developing methods for general terminal time distributions. world health organization (who): who strategic action plan for pandemic influenza situation updates -pandemic (h n ) current who phase of pandemic alert world now at the start of influenza pandemic will vaccines be available for the next influenza pandemic nonpharmaceutical interventions for pandemic influenza international measures centers for disease control and prevention: intreim pre-pandemic planning guidance: community strategy for pandemic influenza world health organization (who) writing group: non-pharmaceutical interventions: their role in reducing transmission and spread mitigation strategies for pandemic influenza in the united state the effect of public health measures on the influenza pandemic in u.s. cities nonpharmaceutical influenza mitigation strategies, us communities, - pandemic transmission dynamics of the etiological agent of sars in hong kong: impact of public health intervention prevention: use of quarantine to prevent transmission of severe acute respiratory syndrome -taiwan novel h n flu: cdc response targeted social distancing design for pandemic influenza a 'smallworld-like' model for comparing interventions aimed at preventing and controlling influenza pandemics comparative estimation of the reproduction number for pandemic influenza from daily case notification data reducing the impact of the next influenza pandemic using household-based public health interventions transmission dynamics of the great influenza pandemic of containing pandemic influenza with antiviral agents strategies for mitigating an influenza pandemic containing pandemic influenza at the source are we ready for pandemic influenza transmissibility of pandemic influenza a bayesian mcmc approach to study transmission of influenza: application to household longitudinal data earn djd: dynamical resonance can account for seasonality of influenza epidemics transmission of influenza: implications for control in health care settings transmission dynamics of severe acute respiratory syndrome danchin a: a double epidemic model for the sars propagation appropriate models for the management of infectious diseases uncertainty in predictions of diseases spread and public health responses to bioterrorism and emerging diseases emergency response to a smallpox attack: the case for mass vaccination smallpox transmission and control: spatial dynamics in great britain poxy models and rash decisions optimal intervention for epidemic models with general infection and removal rate functions optimal control of some simple deterministic epidemic models optimal quarantine programmes for controlling an epidemic spread optimal immunization rules for an epidemic with recovery on the optimal control of a deterministic epidemic optimal control of deterministic epidemics dynamic programming and optimal control athena scientific introduction to optimal control theory springer optimal control and viscosity solutions of hamilton-jacobi-bellman springer what is the best control strategy for multiple infectious disease outbreaks sensitivity and uncertainty analysis of complex models of disease transmission: an hiv model, as an example uncertainty and sensitivity analysis of the basic reproductive rate: tuberculosis as an example modelling an outbreak of an emerging pathogen pre-publication history the pre-publication history for this paper can be accessed here an optimal control theory approach to non-pharmaceutical interventions the authors thank the indiana state department of health for supporting several pandemic planning projects that led to this research. the authors are also grateful to the editors and referees for very insightful and helpful comments. authors' contributions lf conducted the research, including model design, acquisition of data, analysis and interpretation of data, and manuscript drafting. km provided important guidance in model design and methods. he also revised the manuscript critically for important intellectual content. ml supervised the study. he had actively involved in model design and interpretation of data. he also revised the manuscript critically for important intellectual content. all the authors have given approval of the version to be published. the authors declare that they have no competing interests. key: cord- -o uk if authors: tsay, calvin; lejarza, fernando; stadtherr, mark a.; baldea, michael title: modeling, state estimation, and optimal control for the us covid- outbreak date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: o uk if the novel coronavirus sars-cov- and resulting covid- disease have had an unprecedented spread and continue to cause an increasing number of fatalities worldwide. while vaccines are still under development, social distancing, extensive testing, and quarantining of confirmed infected subjects remain the most effective measures to contain the pandemic. these measures carry a significant socioeconomic cost. in this work, we introduce a novel optimization-based decision-making framework for managing the covid- outbreak in the us. this includes modeling the dynamics of affected populations, estimating the model parameters and hidden states from data, and an optimal control strategy for sequencing social distancing and testing events such that the number of infections is minimized. the analysis of our extensive computational efforts reveals that social distancing and quarantining are most effective when implemented early, with quarantining of confirmed infected subjects having a much higher impact. further, we find that “on-off” policies alternating between strict social distancing and relaxing such restrictions can be effective at “flattening” the curve while likely minimizing social and economic cost. www.nature.com/scientificreports/ resource availability and the extent to which social distancing is feasible. among works of this type, djidjou-demasse et al. investigated the optimal control of a single "intervention" input variable for an ode model. moore and okyere formulated a similar problem with additional inputs, including hospitalization rates and environmental spraying. both studies solved the optimal control problem using an iterative forward-backward sweep method and assumed the model parameters to be fully known. while optimal control of epidemiological models has been well-studied (e.g., biswas et al. , neilan and lenhart ), a limiting factor for implementing modeling and optimization concepts during early stages of an outbreak is that obtaining accurate estimates of key model parameters can be challenging. in this work, we report a novel and complete dynamic optimization-based approach to the entire epidemiological modeling and outbreak control workflow for the us covid- outbreak. we first formulate a dynamic optimization strategy for identifying both the values of time-invariant parameters and the historical trajectories of time-varying parameters (i.e., inputs) of an epidemiological model. we then investigate how optimal control of the inputs of the same model, which reflect social distancing and testing, relates to infection mitigation strategies. the optimal control problem is cast as a simultaneous dynamic optimization problem (i.e., reformulated as an algebraic problem) , enabling the natural use of deterministic global optimization technology . this in turn provides solutions that are proven to be globally optimal, whereas iterative schemes such as the above often only guarantee global optimality under certain conditions (the term "global" here refers to the notion that the best possible disease control policy is identified from a set of "local" solutions that otherwise satisfy optimality conditions. "global" in this context should not be interpreted as "world-wide"). furthermore, we show how state estimation should be used to update important hidden model states (e.g., the number unconfirmed infections) to reduce the impact of model inaccuracy. implementations of the parameter estimation and optimal control problems in open-source software are provided freely at https ://githu b.com/balde a-group /covid - . mathematical modeling. we consider the compartmental model shown in fig. , which brings a few key modifications to the conventional seir (susceptible-exposed-infectious-recovered) structure. importantly, the a(t) state is added to account for infected subjects that are not included in the count of confirmed cases, either because they are asymptomatic or because of insufficient testing. the virus is thought to be asymptomatic in - % of cases , and may be transmitted by asymptomatic carriers . we also include a p(t) state, which tracks the population that perishes due to the virus. the modified seair model has six compartments/states: s(t) represents the number of subjects that are susceptible to infection, e(t) the number that have been exposed to the virus, a(t) the number that are infected but asymptomatic/unconfirmed, i(t) the number with confirmed infections, and r(t) the number that have recovered from infection. the states are assumed to sum to a known total population n, and p(t) can be calculated algebraically at all times. note that n is assumed to be constant. this assumption is suitable since the number of deaths is much smaller than the initial population, and thus considering a time-varying total population would have little impact on our model at the expense of increased complexity. the model structure is shown in fig . the seair model is described by the following equations: www.nature.com/scientificreports/ where α a (t) and α i (t) are the rates of exposure to the virus from the population of asymptomatic/unconfirmed and confirmed infected subjects, respectively. these two rates of exposure are defined as time-varying and independent model inputs to reflect different measures taken during the course of the pandemic. specifically, α a (t) corresponds to exposure from asymptomatic carriers (a) and reflects social distancing and/or shelter-in-place strategies. on the other hand, α i (t) corresponds to exposure from infected subjects (i) and reflects quarantining of infected subjects. we also model κ(t) , or the rate at which unconfirmed cases become confirmed, as a timedependent input to reflect varying levels of screening and testing. the other parameters are assumed to be constant over the time horizons considered herein. t − latent is the inverse of the latent period of the virus, or the time before an exposed subject becomes infectious. we assume a value of t − latent = . days − based on peng et al. the parameter ρ describes the infectious period for subjects with unconfirmed infections, for which we assume a value of ρ = . days − , based on rocklöv et al. the rates at which subjects with confirmed infections recover and perish are described by β and µ , respectively. finally, γ describes the rate at which recovered subjects become susceptible to the disease again. since the immunity period for the virus is unknown, we assume a value of γ = . while these parameters largely describe the virus itself, they may change over longer time horizons, e.g., as new treatments are developed. a potential future avenue of research is to consider parameters β and γ to be time-varying, to reflect decision-making regarding budget allocation for medical supplies and equipment, as well as health care capacity expansion over time. in summary, the seair model ( )-( ) has three time-varying inputs ( α a (t) , α i (t) , and κ(t) ), three parameters ( t − latent , ρ , and γ ) whose values are based on available literature, and two parameters whose values must be estimated ( β and µ ). note that since e(t) and a(t) are unmeasured, their magnitudes relative to the infected population i are effectively set by (the bounds on) κ . as the prevalence of asymptomatic infection is established (e.g., bendavid et al. estimate up to % prevalence in santa clara county using serological testing) the magnitudes of e and a may be adjusted by scaling κ , and other parameters as relevant. parameter estimation results. this work primarily addresses the current covid- situation in the us, but the still relatively early stage of the outbreak in the us renders the available data insufficient to fit parameters reflective of prevention measures already in place. for this reason, we perform a comparative analysis by solving the parameter estimation problem for italy, spain, and germany, where, while the epidemic has not been fully contained yet, the daily number of new confirmed cases has been on a steady decline. model parameters for the us, italy, spain, and germany. figure shows the predicted values obtained by solving the parameter estimation problem and the historical data by country, retrieved by the center for systems science and engineering (csse) at johns hopkins university (https ://githu b.com/csseg isand data/covid - ; accessed april , ). day of the dataset corresponds to january , while day corresponds to april . the solid line in each state trajectory plot shows the mean predicted value. standard deviations for the predictions were estimated using bootstrapping, with the time-invariant parameters sampled from a multivariate normal distribution. the means and covariances of the estimated parameters for each country are provided in the supplementary information. plots corresponding to infected, recovered and perished (left plots in fig. ) were obtained by simulating ( )-( ) using the estimated trajectories of the time-varying inputs, shown in the right-most column of fig. . parameter estimation results provide several interesting insights. first we note that the mortality rates, while higher for the european countries, are comparable in magnitude for the four countries considered and are similar to prior estimates . the higher death rates in italy and spain are likely explained by demographic factors (e.g., age), as well as the medical resources available to treat the infected population. however, the simulated trajectories of p in italy and spain deviate slightly from the historical data, especially around day (fig. ) , suggesting that the death rate µ may be time-varying. while µ can vary in reality due to improved treatment, early detection bias, etc., it is difficult to anticipate future changes, and we therefore approximate it as a constant. from the trajectories of α a , α i , and κ , we observe that social distancing measures (i.e., lower values of α a ) were first implemented in europe, and more recently the us, which agrees with their true chronology. this insight is confirmed by the fitted values for e , with italy having the highest value and the us the lowest, which is representative of the beginning of the outbreak in each country. in general terms, the identified evolution of containment and testing measures follow similar trends for all four countries, which supports that the model www.nature.com/scientificreports/ structure is correct and can appropriately reflect (see "methods") different dynamics of the same underlying disease. further, we expect that the parameters obtained for the us, while being fitted using relatively premature information, are likely an adequate representation of the current covid- situation. for the estimated parameter values as described previously, we simulate the results of implementing two different simplistic control policies: (i) continuing with strict social distancing, quarantining, and testing, policies that result from continuing to lower the asymptomatic ( α a ) and infected ( α i ) exposures shown in fig. ; and (ii) a relaxed policy with more lenient measures and reduced testing, in this case the values of α a and α i are increased to . and . , respectively, while κ is decreased to . . the population levels resulting from implementing these two policies are shown in fig. , where the numbers of recovered subjects are omitted for the sake of brevity. we additionally show the number of new confirmed cases per day, as it is a commonly used metric to illustrate the current spread/containment of the virus. since the three european countries showed very similar trends under the two policies considered, we only compare the results for the us to those obtained for italy. on the one hand, from fig. it is evident that relaxing current control policies can result in an alarming number of infected cases and deaths, particularly in the us. on the other hand, continuing with the strict shelter-in-place measures and maximizing testing can result in earlier and substantially flatter pandemic peaks, with significantly lower numbers of casualties. while the latter approach is the most effective at preventing further exponential spread of the disease, it is also the most socially and economically disruptive policy. in light of this trade-off, we argue that by means of employing optimal control concepts it is possible to find effective policies that maintain the number of infected cases below a given threshold, while minimizing the extent of social and economic disruption. here, we consider the optimal control of the covid- infection in the us using the deterministic seair model ( )-( ) with the mean values of the time-invariant parameters as described above. as such, the optimization problem only considers the mean predicted values (e.g., solid lines in figs. and ). future work may consider a stochastic optimization approach that, at the expense of increased computational effort, leverages information about model uncertainty. optimization of future actions. we solve a dynamic optimization problem that minimizes a measure of socioeconomic cost, subject to keeping the peak (max) value of the infected population below a given number i peak . the lower bound of κ(t) is raised to . , from . in fig. www.nature.com/scientificreports/ figure depicts the solution of this problem for the cases where i peak = , subjects and i peak = , , subjects. the optimization problem was solved for the days following the end of the available dataset ( t = , t f = ), with the initial condition at t computed by simulating days - . although the optimization problem only considers the mean values (solid lines), two standard deviations are shown in the shaded areas to reflect parametric model uncertainty for the given inputs, computed using the same bootstrapping approach as above. considering the current status of the pandemic in the us, keeping the peak below , infected subjects is very challenging. note that the minimum feasible value of i peak corresponds to a peak of , infected subjects. therefore, maintaining i(t) ≤ , requires immediately decreasing α a and α i to their lower bounds for approximately the next days, during which the exposed population can decrease significantly. after this period, α a is relaxed to its upper bound for seven brief periods (fig. , left) . however, α i remains at its lower bound at all times, reflecting a strict quarantining policy. this policy reveals that the impact of α i on the infected population size is larger than that of α a . in turn, this suggests that quarantining of infected people is more important than social distancing, which mitigates exposure to unconfirmed cases. the optimization problem penalizes κ to account for the cost of testing, and κ is decreased during periods of decreased α a , suggesting that testing is less important during times of social distancing/lockdown. intuitively, when the exposure to asymptomatic subjects is already low due to social distancing, there is less benefit to testing asymptomatic subjects (and transferring them to the similarly quarantined infected population). on the other hand, κ is increased preemptively for each period of increased α a , suggesting that testing is most important in the days leading up to a period of relaxed social distancing. this testing moves asymptomatic subjects to the confirmed-infected population, which can remain quarantined. figure (right) depicts the solution obtained for a value of i peak = , , people. while α i again remains at its lower bound at all times, re-emphasizing the importance of quarantining infected subjects, there are more frequent and longer periods of increased α a . in this case, κ remains at its upper bound for most of the control horizon. in both cases, the optimization problem does not account for the effect(s) of the control policy after days, and growth of the infected population near the end of the time horizon may be concerning. this can www.nature.com/scientificreports/ be addressed, e.g., using a moving horizon control strategy, where policy measures are revised periodically, as discussed below. the lower bound used for α i is lower than estimated values for the us for the past (fig. ), corresponding to a new level of quarantining. furthermore, the current value of α a , or social distancing, may not be economically sustainable over longer periods. to investigate the effect of not achieving these proposed levels, we solve the same problem with i peak = , , people, using different lower bounds for the inputs. figure shows the optimal optimal containment and testing strategies to limit peak infections to , , in the next days for different constraints on α a and α i . top: α a (t) . bottom: κ(t) . the shaded grey area indicates the solution found using the normal bounds, replicated from fig. www.nature.com/scientificreports/ input profiles found for the cases where the lower bound of α a is doubled from . to . (dotted blue lines), and where the lower bound of α i is doubled from . to . (solid black lines). when the lower bound of α a is doubled (less social distancing is achieved during lock-down periods), the same infected population peak can be maintained by increasing the frequency of social distancing periods. however, when the lower bound of α i is doubled (less quarantining is achieved), maintaining the same number of peak infections requires social distancing at almost all times, closely resembling the control policy found for a lower peak (fig. , left) . in either case, the optimal value of α i remains at its lower bound (either . or . ) at all times and is therefore not shown. periods of social distancing are further increased in both frequency and duration if both bounds are doubled. compared to the decreases seen in fig. for the cases of continued control, the solutions in fig. seek to merely flatten the growth of the infected population. the associated optimal control policies resemble "bangbang" control (the inputs are always at the lower or upper bound), which can be expected from a systemstheoretic point of view for ( )-( ), as the equations are linear functions of the inputs α a , α i , and κ . this result supports the strategy proposed by the imperial college covid- response team , which involves periodic suppression measures. their strategy triggers the start of a social distancing period by when the number of weekly icu cases increases past an "on" threshold, and the end of the period when the same number decreases below an "off " threshold. figure shows that this type of strategy maintains a low exposed population, and therefore flattens the growth of the infected population while still allowing periods of social mobility. revisiting past actions. we examine the same optimal control problem with a time horizon starting from day (i.e., covering days in the past). rolling the time horizon of the optimization problem backwards to t = , allows us to investigate the optimal inputs for days - , corresponding roughly to the latter half of march and the first half of april. figure (left) depicts the solution obtained for a value of i peak = , people. the initial condition at t = was computed by simulating days - . the minimum historical values for α a and α i were used as their respective lower bounds for days - , such that the optimal control policy for days - only involves an extent of quarantining and social distancing already experienced by the general population. in this case, maintaining the number of infected subjects below , appears easy. the minimum feasible peak of infected subjects is , , compared to , when the optimization starts at day . therefore, maintaining i(t) ≤ , can involve extended periods of no social distancing α a = . . similar to the result in fig. (right) , testing and quarantine are important in this scenario: κ remains at its upper bound and α i its lower bound. the solution for an order-of-magnitude lower minimum peak, corresponding to , infected subjects is shown in fig. (right) . the optimal control policy for this scenario involves more frequent periods of social www.nature.com/scientificreports/ distancing. early implementation of testing and quarantining strategies clearly has an enormous effect, manifest in the large difference between the historical inputs (dashed) and optimal inputs (solid) in days - of fig. . estimation of hidden states and moving horizon control. the seair model ( )-( ) includes two hidden states, e(t) and a(t), which are not measured in practice (note that widespread serological testing may eventually reveal the true levels of the asymptomatic population a). nevertheless, solutions for the optimal control problem based on ( )-( ) are highly dependent on the values of e(t) and a(t), and therefore the initial conditions e(t ) and a(t ) . fig. (left) depicts the optimal control policies for i peak = , , people over the next days (similar to fig. , right) , where e(t ) and a(t ) are now underestimated by a factor of three. this is a practically motivated scenario, as the number of asymptomatic cases is considered largely uncertain , . the dashed-dotted lines show the state profiles predicted by the optimization problem (with incorrect initial conditions), while the solid lines show their true evolution for the given inputs. the error in the hidden states e(t) and a(t) causes the predicted and actual profiles to diverge over time. by the end of the days, there are . million actual infected subjects, almost % greater than the predicted . million. to account for discrepancies between the modeled and true systems, the optimal control inputs should be periodically updated. we propose to achieve this with a moving horizon decision-making strategy: after a given length of time, the dynamic optimization problem is re-solved, setting the measured values of the states as their initial conditions. the values of the un-measured, hidden states can be estimated using state estimation techniques, such as the kalman filter (the optimal linear estimator) or its nonlinear extensions. we consider a relatively long time span of days before each revision of policy decisions (and re-optimization), as planned suppression strategies likely cannot be altered quickly. figure (right) shows the optimal trajectories found for the same problem (initial conditions for e and a underestimated by a factor of three) using the moving horizon strategy. the vertical dotted lines indicate times when the optimization problem was resolved. values of the hidden states were estimated daily with the (discrete) unscented kalman filter , implemented using the pykalman library (https ://pykal man.githu b.io/; accessed april , ). measurements for state estimation and control updates were simulated by adding independent, normally distributed noise to the true state values. a standard deviation of , people was used for the measured states (i, r, and p) based on the size of residuals during parameter estimation. the predicted values (dash-dotted lines in fig. ) are first updated when the optimization problem is re-solved at t = days. the solutions to the re-optimization problems are shown in the supplementary information. the figure . optimal moving horizon control policy (right) to limit peak infections to , , , with e and a underestimated by a factor of three at t = , and comparison to the same situation without a moving horizon strategy (left). top: predicted (dash-dotted) and true (solid) population numbers. bottom: containment and testing profiles. the shaded grey area indicates past days, which were simulated using historical inputs (not optimized). the policies are updated every days with daily state estimation. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ effect of this moving horizon strategy is quite significant, in comparison to the optimal policy shown in fig. (left). periods of social distancing are quickly increased after day to account for the larger-than-expected increase in infected population. testing levels, reflected by κ , are also updated accordingly. the moving horizon approach exhibits three clear benefits: • errors between the model predictions and reality are minimized by incorporating new measurements as they become available • the infected population does not grow exponentially at the end of the time horizon, as is the case when considering only a single time horizon. this work investigated dynamic optimization strategies to characterize and control the us covid- outbreak, by minimizing the socioeconomic cost associated with containment strategies and testing. the results provide several overarching conclusions. the quarantining of infected subjects is the most important of the considered mitigation strategies and should be maximized at all times. additionally, periods of social distancing/lock-down help to flatten the peak by preventing exposure from asymptomatic and unconfirmed cases. screening and testing for the disease are key immediately preceding periods of relaxed social distancing, in order to minimize the number of unconfirmed infections during periods of social mobility. early action has much larger effects than later interventions, even as the later interventions are more drastic. optimal policies are highly dependent on estimates of "hidden states, " i.e., the asymptomatic and unconfirmed cases. moving horizon (periodically updated) policies and state estimation should be used to mitigate inaccuracies in the model and counts of asymptomatic/unconfirmed cases, by accounting for new data as they becomes available. the "on-off " policies identified as optimal are characteristic of the class of problems considered (linear objective function and input-affine nonlinear model). further, these policies are likely the easiest to implement in practical scenarios and to convey to the general population (as opposed to a policy where the social distancing parameters would take values between their upper and lower bounds). their implementation would simply alternate between the strictest possible limitations, followed by periods of relative freedom of movement. the frequency of these periods may be restricted, e.g., by including rate-of-change constraints in the optimization problem, to account for the ability of a country or local authority to intermittently enforce social distancing policies. the model used in this work does not include population influx to and/or outflux from the given system. therefore, the optimal containment and testing strategies found do not account for new cases that may enter from outside the us. we also assumed that the recovery and death rates for the virus are constant and equal to their historical values for the next days. thus the results do not account for the possibility of, e.g., improved medical treatments, vaccine development, and/or viral mutation. similarly, the model does not account for possible surveillance/detection bias in the historical data (e.g., increased likelihood of testing for subjects with more severe symptoms). the impacts of these assumptions are topics for future work. also, while sameni determined fixed points and did linear stability and observability analyses for a similarly-structured seir model with timeinvariant parameters, another topic for future work is the extension of this mathematical analysis to the seair model with time-dependent parameters used here. parameter estimation. we follow nonlinear (least-squares) regression for parameter estimation, which can be cast as an optimization problem, where the objective is to find the parameter values that minimize the mean squared error (mse) between the predicted states and their measured values. the measured values correspond to data retrieved by the center for systems science and engineering (csse) at johns hopkins university (https ://githu b.com/csseg isand data/covid - ; accessed april , ) and are provided in the supplementary information for several countries. the data consist of the total number of infected ( î j ), recovered ( r j ) and dead ( p j ) reported subjects, where j represents each day during the time period from january to april , where the index j represents the time period each value was recorded. the mse is given by: we note that when solving the parameter estimation problem, the contribution of the term ρa(t) to the recovered state r(t), is removed from the system ( )- ( ) . this is because the data obtained only accounts for those www.nature.com/scientificreports/ recovered that were confirmed to have the disease, meaning the only contribution to the observed recovered is the term βi(t). the parameters with values/trajectories to estimate are α a (t) , α i (t) , κ(t) , β , and µ . furthermore, we must estimate the initial condition of the unreported states e and a at t = . we assume that there are initially no asymptomatic infections. the "seed" of the outbreak is therefore the initial number of exposed subjects e = e(t = ) , which we include as a parameter in the least-squares problem. we include bounds on the possible parameter values, based on values reported for similar models fitted to data from other regions . note that, while κ reflects the level of testing, it also affects the predicted asymptomatic ratio, which cannot be controlled. therefore, tighter bounds should be used for κ than for α a and α i . the estimated parameters and their bounds are summarized in fig. . to prevent over-fitting the available data, we restrict the time-varying inputs ( α a , α i , and κ ) to be piece-wise constant over five-day intervals. these constraints reflect the fact that policies implemented have significant time delays in steering the states towards the desired values, and therefore should not and practically cannot be manipulated too frequently before their effect is observed on the population. additionally, we constrain α a (t) and α i (t) to be monotonically decreasing over time, reflecting the enforcement of increasingly stricter disease control measures. similarly, κ(t) was constrained to be monotonically increasing over time to account for increased covid- screening availability. to easily interface with raw data sources, the least-squares regression problem was implemented in python using the pyomo modeling and optimization package . we discretized the dynamical system ( )-( ) with respect to the time domain using orthogonal collocation on finite elements , with one finite element per day. we used ipopt to solve the resulting nonlinear dynamic optimization problem to local optimality. standard deviations for the time-invariant parameters were approximated by fixing the time-varying inputs to their estimated trajectories, and solving a maximum likelihood problem for the time-invariant parameters using the model validation tool in gproms (general process modeling system) v . . . residuals were assumed independent and normally distributed, with variances estimated in the same maximum likelihood problem. the parameter covariances (see supplementary information) are then computed from the diagonal entries of the hessian of the objective function. optimal control. the aim of the optimal control problem is to find the trajectories of "handles" α a (t) , α i (t) and κ(t) , that minimize (or maximize) the value of a certain objective function. most response measures for the covid- outbreak seek to "flatten" the epidemic curve, that is, to contain the growth rate of the number of infected subjects via a combination of social distancing and testing. clearly, social distancing and isolation/ quarantining carry significant social and economic costs. we thus formulate the optimal control problem as a dynamic optimization problem, aiming to minimize a measure of social and economic cost subject to ensuring that the maximum number of infected subjects remains under a given peak value, i peak . the optimization problem is expressed mathematically as: where c(t) is the cost function, and κ is the relative cost of testing (increasing κ ). testing costs are assumed to be relatively small in comparison to isolation measures, and we selected κ = . . while the current cost function equally penalizes decreases in α a and α i , an additional weighting factor could be introduced to distinguish between the socioeconomic costs of social distancing and quarantining. the solution to ( ) can provide control policies over the full time horizon [t , t f ] as in the case of the initial studies presented in this work. alternatively, the policies can be updated after a shorter horizon (before t f is reached), as in the case of the moving horizon approach. in the latter case, the time window considered, [t , t f ] is shifted forward at a pre-determined time interval. note that t f is typically longer than the frequency at which the optimization problem is recomputed, and only the solution for the first time step(s) is implemented. for example, the moving horizon scenario presented here is solved with t f = days, but with policies updated after each -day period. while the problem ( ) identifies the minimum societal cost required to achieve a certain peak value of infections, a related, but different, problem could be formulated by minimizing i peak subject to an upper bound on c(t), i.e., finding the lowest achievable peak for a given total societal cost. for this study we investigate the relationship between c and i peak by solving ( ) for varying values of i peak , provided in the supplementary information. the optimization problem ( ) was again solved by discretization of the time domain using orthogonal collocation, with one finite element per day. to report the best possible solutions, the resulting algebraic optimization problem was implemented in gams and solved using the commercial global optimization solver baron v . . . problems were solved to a . % optimality gap (i.e., the reported solution is proven to have an objective function value within . % of that of the best possible solution). we additionally provide an implementation www.nature.com/scientificreports/ the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? world health organization declares global emergency: a review of the novel coronavirus (covid- ) lockdown in italy: personal stories of doing science during the covid- quarantine evaluation of the lockdowns for the sars-cov- epidemic in italy and spain after one month follow up the effect of human mobility and control measures on the covid- epidemic in china estimated effectiveness of symptom and risk screening to prevent the spread of covid- modeling infectious diseases in humans and animals data-based analysis, modelling and forecasting of the covid- outbreak epidemic analysis of covid- in china by dynamical modeling 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systems enterprise. general process modeling system (gproms a polyhedral branch-and-cut approach to global optimization models are available at https ://githu b.com/balde a-group /covid - .received: april ; accepted: june acknowledgements partial financial support from the national science foundation (nsf) through career award is acknowledged with gratitude. the authors also thank dr. richard pattison (apeel sciences) for his insightful comments throughout this study. all authors conceived the experiments; c.t. and f.l. implemented the optimisation models, conducted the computational experiments, and analysed the results; all authors prepared and reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - kmicf authors: bonyah, ebenezer; badu, kingsley; asiedu-addo, samuel kwesi title: optimal control application to an ebola model date: - - journal: asian pac j trop biomed doi: . /j.apjtb. . . sha: doc_id: cord_uid: kmicf ebola virus is a severe, frequently fatal illness, with a case fatality rate up to %. the outbreak of the disease has been acknowledged by world health organization as public health emergency of international concern. the threat of ebola in west africa is still a major setback to the socioeconomic development. optimal control theory is applied to a system of ordinary differential equations which is modeling ebola infection through three different routes including contact between humans and a dead body. in an attempt to reduce infection in susceptible population, a preventive control is put in the form of education and campaign and two treatment controls are applied to infected and late-stage infected (super) human population. the pontryagins maximum principle is employed to characterize optimality control, which is then solved numerically. it is observed that time optimal control is existed in the model. the activation of each control showed a positive reduction of infection. the overall effect of activation of all the controls simultaneously reduced the effort required for the reduction of the infection quickly. the obtained results present a good framework for planning and designing cost-effective strategies for good interventions in dealing with ebola disease. it is established that in order to reduce ebola threat all the three controls must be taken into consideration concurrently. the principal aim of modeling infectious diseases is to be able to make judicious decisions in the application of control interventions of the infection to eliminate and ideally to eradicate it from the human population. simulations and modeling can optimize control efforts such that limited resources are targeted to achieve the highest impact [ ] . the aim of this paper is to review the epidemiology of the ebola pandemic and discuss the optimal control model that governs the spread of the virus in human population and suggest the optimum control strategies to control and curb the spread in the future. the world witnessed an unprecedented ebola outbreak in west africa which in the end was reported in some parts of europe and north america [ ] . by december th there had been confirmed reported cases in excess of in total with over people losing their lives particularly in west africa and to a lesser extent elsewhere in the world. outside africa; italy, spain, the united kingdom and united states of america [ ] were also affected albeit with no case fatalities except for one in the usa ( table ) . the outbreak has been acknowledged by the world health organization as a public health emergency of international concern. the three west african countries (guinea, sierra leone and liberia) by far been hardest hit now account for about . % of all infections and deaths. these countries are known to have only recently emerged from long periods of conflict and instability and thus have weak health systems, human and infrastructural resources [ ] . ebola is known to be transmitted to humans via contact with bodily fluids and secretion of infected animals mainly fruit bats, monkeys, porcupines, forest antelope and the like. it is thereafter spread from person to person through direct contact with infected persons [ , , ] . the incubation period beyond which infected people become symptomatic has been estimated to range between and days [ ] . the ebola virus is a unique virus having a filamentous, enveloped non-segmented negative-sense rna. it belongs to the genus ebolavirus, within the family of filoviridae. its envelope glycoprotein facilitates the entry of the virus to living cells [ , ] . till date five strains of the virus are known: zaire, sudan, tai forest, bundibugyo, and reston with zaire stain being the most virulent with up to % fatalities [ ] . the pasteur institute (lyon, france) has sequenced the viral strain currently under circulation in west africa and reports a strong homology of % with zaire ebolavirus (the most virulent). the origin of the ebola virus has been somewhat unclear [ , ] . however, in , leroy and co-workers reported in the nature, evidence of asymptomatic infection by ebola virus in three species of fruit bat [ ] . this indicated that fruit bats belonging to the family pteropodidae are the natural host of the ebola virus. ebola viral disease has no effective treatment or vaccines, currently only supportive care can be given to patients. emerging tropical infectious diseases have been persistent in causing untold economic hardships to relatively poor countries with weak health systems. the overarching goal of public health is to reduce disease burden by curtailing transmission or mitigating its severity. there are at least two fundamental public health guiding principles that exist to manage the spread of an infectious disease like ebola viral disease that has no effective vaccines or treatment. these are (i) effective isolation of persons with symptoms and (ii) tracing the contacts of symptomatic cases for clusters of exposed persons and quarantining them for monitoring [ ] . mathematical models have played a vital role in the dynamics and control of many epidemics including malaria, severe acute respiratory syndromes and ebola [ ] . some previous models of ebola virus, especially the predictive models endeavour to calculate a threshold called basic reproductive number r . the dynamics of transmission of the disease has been analyzed in terms of the reduction of the basic reproductive number [ ] [ ] [ ] [ ] . however, all these models fail to take into account time dependent control strategies and all their discussions have been concentrated on prevalence of the disease at equilibra. time dependent control has been employed in the study of dynamics of diseases. for example, rachah and torres, investigated the effect of vaccination on a proportion of susceptible population and observed that the rate of infection of ebola reduced due to this intervention [ ] . chowell and nishiura also applied time optimal control to study ebola epidemic reduction [ ] . other studies on using time optimal control to provide appropriate interventions to minimize the spread of diseases have also been carried out successfully [ ] [ ] [ ] [ ] . this technique of studying control strategies present an enviable theoretical results that can assist in providing tools for designing epidemic control programmes. in this work, time dependent optimal control is explored and considered which deals with both "case holding" and "case finding" on ebola model proposed by rivers et al. [ ] . the model assumes that there is a difference between first-stage infections and late-stage infection called super infection. their model further assumes individuals in the latent stage develop active ebola infection at a given rate. it also assumes that a proportion of both first and late stage-infection (super infection) recover and others move to death compartment. we present three control mechanisms which comprise two "case finding" and a "case holding" in the model. the "case finding" is usually made up of activities that lead to preventive measures including screening, public education and others. the "case holding" also has to do with designed activities that ensure patients take their drugs within stipulated times so that they are cured. the first case finding is incorporated by adding a control term that characterizes the contact between susceptible and infectious individuals so that the rate of infection will be reduced. the second case finding is instituted in the model by adding a control term that identifies proportions of those individuals in the latent stage or exposed to the disease and cure them so that the rate of getting the disease will be reduced. the holding is incorporated in the model by adding a control term that may minimize the treatment failure rate of individuals with ebola disease. we choose the reduction of infected individuals of ebola to be our main objective having a lower cost of the controls. the paper is arranged as follows: section is devoted to describing an ebola model with three control terms incorporated. in addition, the objective function is introduced in this section. in section , the analysis of optimal control is discussed. section contains some numerical studies of optimal controls. finally, section deals with the conclusions of the studies. the model structure is presented in figure and the state system of the ebola model is the following six nonlinear ordinary differential equations proposed by pontryagin et al. [ ] and slightly modified: figure . stage-structured compartmental model of ebola virus disease, which splits the population into susceptible (s), exposed (e), first-stage infected (i ), late-stage infected (i ), recovered (r), and funeral transmissible (f). red compartments are transmissible, and recovery rates are greater from i than from i [ ] . d denotes the fraction of infected who progress to second stage of infectious and d is the fraction who subsequently progress to death. the natural per capita mortality of ebola disease rate is denoted by m. the control functions being employed, u (t), u (t) and u (t) are bounded lebesques integrable functions. the "case finding" control u (t), deals with efforts that facilitate the keeping of a distance between susceptible and infectious individuals including education and public campaigns. the "case holding" control, u (t) deals with effort needed to identify the proportion of typical ebola exposed individuals that is known and will be put under treatment in order to reduce the number of individuals that may turn to be infectious. the term u (t) deals with the effort that ensures those that are infectious both in the first and super infection stages are given treatment and monitored to take their drugs in order to minimize the number of individuals developing and dying of ebola. our goal, therefore, is to minimize the number of infected individuals with the ebola virus while at the same time keeping the cost of treatment very low. in mathematical perspective, for a fixed terminal time t f , the problem is to minimize the objective functional it is assumed that cost of treatments is of nonlinear and takes a quadratic nature. the coefficients, a , a and a , are representing the balancing cost factors which have to do with the size and importance of the three segments of the objective functional. thus, we seek to determine an optimal control, u * , u * and u * , such that where u = fðu ; u ; u Þ l ð ; t f Þ|c i u i d i ; i = ; ; g and c i ,d i ,i = , , , are denoted as fixed positive constants. in the entire work, we have assumed that the total population n to be constant. in order to achieve this, we choose l = mn. the indispensable conditions that an optimal pair must satisfy emanate from pontryagin's maximum principle [ ] . this principle actually does convert ( )-( ) into a problem of minimizing pointwise, h, with respect to u , u and u : g i denotes the right hand side of the ith differential equation of the state variable. by employing pontryagin's maximum principle [ ] and the existence results obtained for the optimal from [ ] , we arrive at theorem . there exist an optimal control u * , u * , u * and associated solution, s * , e * , i * , i * , r * and f * , that minimizes j (u , u , u ) over u. furthermore, there exist adjoint functions, l (t),…, l (t), such that with transversality conditions l i À t f Á = ; i = ; :::; ( ) proof. corollary . fleming and rishel ( ) present the existence of an optimal control owing to the convexity of integrand of j with respect to (u , u , u ), a priori boundedness of the state solutions, and the lipschitz property of the state system with respect to the state variables [ ] . by employing pontryagin's maximum principle, we have ::::::: evaluated at the optimal control and corresponding states, which result in the stated adjoin system ( ) and ( ) [ ] . by considering the optimality condition, vh vu = ; vh vu = ; vh vu = ( ) and obtaining solution for u * , u * , u * gives subject to the constraints, the characterization ( ) are determined at u * on the set ft|c < u * i ðtÞ for i = ; ; g. on this set, owing to the priori boundedness of the state and adjoint functions and the resulting lipschitz structure of the ordinary differential equations, we get the uniqueness of the optimal control for small. the uniqueness of the optimal control does move with the uniqueness of the optimality system, thus ( ) and ( ), ( ) with characterizations ( ) . there is a restraint on the length of the time interval to ensure that uniqueness of the optimality system is obtained. this smallest restrictions on the length on the time interval has to do with the opposite time orientations of ( ) and ( ), ( ) . the state problem presents initial values and the adjoint problem also deals with final values. this restriction is frequently observed in control problem [ , ] . in this section, we investigate the effect of optimal strategy on ebola transmission applying some numerical techniques. the optimal strategy is achieved by obtaining a solution for the state system ( ) and co-state system ( ). an iterative scheme is explored and used to determine the solution for the optimality system. the state equations are initially solved by guessing for the controls over the simulated time applying a forward fourth order runge-kutta scheme. in addition, the co-state equations are at the same time computed by employing a backward fourth order runge-kutta scheme with the transversality conditions. this is then followed by the controls being updated by employing a convex combination of the preceding controls and the value obtained from the characterizations of u * , u * , u * . this process is allowed to go on and iteration is ended if the values of unknowns at the previous iteration are almost the same as the value obtained at present iteration [ ] . for numerical simulation, the state system solution is determined based on forward in time with initial conditions x( ) = ( , , , , , ), whereas the co-state system is also dealt with backward in time. in the light of numerical simulation, we employed the following parameters: d = . /days; a = /days; b = . /days; d = . / days; g f = . /days; m = . /days; g = . /days; g = . /days; b = . /days and the weighting control b = , b = , b = . the weight factor b associated with control u is less or equal to weight factor b associated with control, however, weight factor b associated with control u could be higher than all due to cost associated with it because of the cost implications. with this approach, only the control u which deals with prevention is applied to optimize the objective function j, whereas the control u and u on treatment are set to zero as shown in figure . in order to reduce infection control, u should be sustained intensively for the first seven days. figure shows the optimal solution for first stage infection i and super infection i which depict substantial difference with and without control in both figures. in figures and , it is observed that effective preventive mechanisms such as proper education and campaign will help reduce the rate of infection of ebola in communities. with this scenario, we activate controls u and u on prevention and treatment to optimize the objective function j, whereas the control u which is the treatment of first stage of infection and super infection is set to zero as shown in figure . we can observe in figure that it will require about days intensive intervention in the form of education and campaign and at the same time giving intensive treatment for those exposed to the disease through screening for about days. figure shows the optimal solutions for both first stage infection and super infection. clearly there is a vast difference between without treatment and treatment. therefore, it can be deduced that giving the right education and campaign to susceptible individuals and providing good treatment to those exposed individuals will help reduce epidemic significantly. with this approach, all the controls u , u and u treatments and prevention are activated to optimize the objective function j, and none of the controls is set to zero as observed in figure . in figure , it can be seen that with all the controls activated one requires less effort and time to reduce infection with respect to control u and u . however, one needs about days' intensive treatment for those exposed to ebola. figure depicts optimal solutions for first stage i infection and super infection i . it can be observed that there is a clear significant difference in figure , both first stage infection i and super infection i in terms of with controls and without controls when all the three controls activated. it is therefore envisaged that in order to design a cost effective mechanism for effective interventions, the necessary attention should be given to all the three scenarios at the same time. the optimal control strategy applied to this model for the "case holding" is similar to previous studies by rachah and torres [ ] , who used vaccination as a strategy to reduce the number of infection in the susceptible population in susceptible-infected-recovered (sir) ebola model. this "case holding" strategy again is further supported by rachah and torres in their extension of previous work by comparing optimal effect of vaccination on sir and susceptible-exposed-infectedrecovered ebola models [ ] . the numerical results from their comparison studies indicated that vaccination programme is more effective in susceptible-exposed-infected-recovered ebola model than sir ebola model. in this study, the "case finding" activities include mass education and vigorous campaign which also reduced ebola infections on "case finding" scenario. the numerical results obtained based on "case holding" strategy that is treatment is similar to the study of jung et al. [ ] on optimal treatment of tuberculosis. this therefore, shows that proper medication on right time bases will ensure that those affected with ebola can be cured. the numerical result obtained in this study is new and is not contrary to any previous studies on ebola models. in this work, mathematical model of ebola disease with three possible routes of transmission that include prevention and two treatment measures as optimal control has been examined. by exploring and applying pontryagin maximum principle, condition for optimal control which addressed minimizing the disease in a finite time are derived and analyzed. it could therefore be concluded that the combination of education, screening and giving treatment for exposed as well as treatment for infectious population will be a more effective way of reducing ebola disease in a community. optimal treatment figure . the optimal solutions for first stage of infection (i ) and super infection (i ) via treatment. optimal control 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infection model optimal control of the chemotherapy of hiv optimal control applied to biological models predicting and controlling the ebola infection we declare that we have no conflict of interest. key: cord- -drjj k authors: nenchev, vladislav title: optimal quarantine control of an infectious outbreak date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: drjj k this paper studies the optimal control of an infectious spread based on common epidemic models with permanent immunity and no vaccine availability. assuming limited isolation control and capacity constraints on the number of infections, an optimal quarantine control strategy that balances between the total number of infections and the overall isolation effort is derived from necessary optimality conditions. the specific optimal policy is then obtained by optimizing the switching times of this generalized strategy. in the case of a newly emerged disease, these results can be used in a data-driven receding horizon manner to improve actions as more data becomes available. the proposed approach is applied to publicly available data from the outbreak of sars-cov- in germany. in particular, for minimizing the total number of infections or the number of isolated individuals, the simulations indicate that a sufficiently delayed and controlled release of the lock-down are optimal for overcoming the outbreak. the approach can support public health authorities to plan quarantine control policies. since the first recorded covid- infections in wuhan, china, the disease has spread worldwide. there has been a plurality of attempts to interpret and predict the development of the disease based on public data using first-principles epidemiological [ ] [ ] [ ] , data-driven [ ] , or mixed models [ , ] . based on these predictions, a wide variety of temporal isolation policies have been established in most affected countries. however, evaluating the effectiveness of these responses is notoriously difficult due to the reliability of available data, and since their impact on the outbreak evolution can only be measured with a substantial delay. mathematical analysis of first-principle models of biological systems can provide valuable insight, e.g., on their general properties [ ] , or how to control infectious disease outbreaks [ ] . in the simplest model, the population is divided into the susceptible s , infected i and recovered r groups (therefore called the sir model), and their evolution over time is represented as a set of coupled ordinary differential equations. augmenting the state by exposed e individuals, leads to the so-called seir model. applications of outbreak controls minimizing various different cost functions have been proposed for the sir model, e.g., [ ] [ ] [ ] [ ] [ ] to name a few, as well as heuristically tailored policies [ , ] . although s(e)ir models can be too simplistic for many real outbreaks, they often allow deriving complete analytical solutions, thus providing a foundation e-mail address: vladislav.nenchev@gmail.com for rigorous mathematical results upon which more complex models can be built. an issue of practical concern for many disease outbreaks without an available vaccine, such as for sars-cov- as of june , is minimizing the overall quarantine effort or the final outbreak size, while respecting control and capacity constraints on the current number of infections. to account for quarantine measure (similar to [ ] and [ ] ), the sir model is extended by a quarantined subpopulation q . infected individuals are moved depending on the applied control rate from i to q , where they cannot cause additional infections. then, using the direct adjoining approach [ ] and applying pontryagin's minimum principle (pmp) to the corresponding state-and input-constrained control problem, in this paper it is shown that bang-bang or bang-boundary controls are admitted in a particular sequence to solve the corresponding optimal control problem. to obtain the specific optimal policy, the switching times between the bang-boundary subarcs are determined in an induced optimization problem. analogous results can be obtained for the seir model. upon an outbreak of a previously unknown disease, better model parameter estimates can be obtained as more data becomes available, and the induced optimization problem can be recomputed in a data-driven receding horizon manner to improve actions. the proposed methods are applied to publicly available data until june from the outbreak of sars-cov- in germany. in particular, minimizing the total outbreak size or the total number of isolated individuals are studied. the findings of this work can be used to evaluate if the policies that have been established in practice resemble the optimal policy, and support public health authorities in implementing effective future quarantine strategies. the remainder of this paper is structured as follows. in section , a mathematical statement of the problem is provided. in section , the optimal quarantine policy under isolation control and capacity constraints, and, in section , the data-driven receding horizon control scheme are described. then, in section , the approach is applied based on covid- data from germany until june . finally, the main results and future work are discussed in section . we address epidemics with no vaccine availability, in which the only available control is based on isolation. let the entire population p be divided into three sub-populations: susceptible s ; infected i ; and recovered r . births' and deaths' effects are neglected. the recovered population is to be interpreted as those who can no longer infect others, and, who cannot become reinfected by the disease. this is known as the classic epidemiological sir model [ ] . to include quarantine or isolation control, the model is augmented by introducing a time varying quarantine strength control u ( t ) and a corresponding quarantined (and also recovered from the disease) population q . then, with x = [ s, i, r, q] t ⊆ r + the sub-populations' evolution is governed by the following system of coupled nonlinear ordinary differential equations: where the population p = i = x i (t) , and the infection b and recovery m rates are assumed to be constant in time. note that the model assumes homogeneous mixing among the sub-populations, as well as uniform susceptibility and disease progress for every individual in the population. the sir model assumes a direct transition from susceptible to infected. it can be easily augmented by including exposed individuals e to account for a significant incubation period during which individuals have been infected but are not yet infectious themselves. this so-called seir epidemiological model [ ] has been employed in several prior studies, such as the sars outbreak, e.g., [ , ] , as well as the covid- outbreak [ ] [ ] [ ] . since the seir model did not provide any additional qualitative insights in the studied problem, for simplicity, the sir model is analyzed, and details for the seir model can be found in the appendix. an important characteristic of epidemics is the reproduction number thus, applying the control u ( t ) results in reducing the effective reproduction number r t . determining the maximal quarantine control u max is highly dependent on local quarantine policies and population specifics, directly corresponding to the minimally achievable reproduction number ( ) . another important parameter of epidemics is the fraction α s of severe conditions among infected individuals requiring hospitalization and admission to an intensive care unit. highly infectious diseases (with a high r t ) causing many severe conditions in the infected population (corresponding to a high α s ), may quickly lead to an overload of available treatment resources and, thus, cause a large number of additional potentially avoidable deaths. assuming that the overall available capacity is described by α c , this capacity limitation can be described as the in this work, the goal is to obtain an optimal quarantine control policy u ( t ), t ∈ [ , t f ] for a fixed final time t f , that minimizes a weighted combination of the total number of infections and the overall number of quarantined individuals at time t f . note that the overall number of quarantined individuals corresponds to the applied level of containment and can, thus, be linked to the economical impact of the outbreak. the total number of infections is denoted by the sum of the final recovered and final quarantined populations, and is directly correlated to the overall number of deaths. this can be captured by the cost function where w ∈ [ , ]. the control is limited to u ( t ) ∈ [ , u max ] at all times, such that u = corresponds to an uncontrolled epidemics evolution, and u = u max to the case, when maximal possible isola- t f ] exists, this leads to the following (mayer type) optimal control problem. ( ) is minimized subject to the dynamics ( ) , while respecting the constraint ( ) at all times. in the following, using the direct adjoining approach and applying pontryagin's minimum principle, necessary optimality conditions are derived for the optimal control problem above. further, it is shown that the obtained locally optimal solutions constitute an optimal generalized strategy under certain assumptions. as ( ) is a pure state constraint, (higher order) time derivatives are used to obtain the control u ( t ) that yields a system evolution along the active state constraint. since u ( t ) appears linearly in the state equation of x ( ) , the first total time derivative of the function h ( x ( t )) ( ) contains the control explicitly: . if τ and τ are maximal, let τ be the entry-time and τ the exit-time of the boundary arc. on the boundary arc, thus, the control on a boundary arc is obtained from the boundary control ( ) depends on x and is, therefore, not guaranteed to be in the feasible control set [ , u max ] . thus, assume that to derive first order necessary optimality conditions by applying pmp, a lagrange multiplier η(t) ∈ r associated with the state constraint ( ) is introduced. for that, based on the aforementioned regularity and boundedness conditions on a boundary arc, and supposing that the state space constraint is not active at the initial and final time, i.e., h ( x ( )) < , h ( x ( t f )) < , the direct adjoining approach [ ] is applied. the corresponding augmented hamiltonian is given by with the adjoint variable λ ∈ r . as shown in maurer et al. [ ] , there exist an absolutely continuous λ( t ) and a piecewise absolutely continuous multiplier function η( t ), such that the following for the sir model, the following holds: as the hamiltonian ( ) is linear in u ( t ), the optimal control will depend on the sign of the switching function, given by as suggested in ledzewicz and schättler [ ] , with the total time derivative of ( ) and the costates ( ) , holds on a boundary arc with t ∈ [ τ , τ ]. this yields the explicit expression for the multiplier assuming that a solution of problem exists and the control u ( t ) has finitely many switching times, the following proposition holds. the generalized optimal control policy for problem is if t > t , the tangency constraints ( ) must also hold. proof. on interior arcs, where the state constraint is inactive h ( x ( t )) < , from the minimum condition ( ) , the optimal control is the switching function ( ) is zero ⇐⇒ λ (t) = or x (t) = . the latter case is trivial and will be neglected in the following. to fulfill λ (t f ) = , λ ( ) > and ˙ λ ( +) ≤ , such that the optimal control policy may start with a zero segment for [ t , t ). if ) > and σ (t −) < assuming u (t −) = , and as −˙ σ (t )( − u max ) > , a second segment for t ∈ [ t , t ) may have the control u max . on a boundary arc, given the regularity and boundedness conditions for the boundary control, the minimum condition ) ≤ u max (from the boundedness condition of the boundary control) and ˙ x (t +)( ū b ) = , and thus, the continuity conditions on the boundary σ ( substituting relevant equations into ( ) yields , resulting from the boundedness condition of the boundary control, completes the additional tangency constraints ( ) . if h ( x ( t )) < and σ (t) = for t ∈ [ ˜ τ , ˜ τ ) , a singular arc may occur. since ∂ σ /∂ u = holds trivially, the singular control u s can be obtained from d σ /d t = with η(t) = and λ (t) = , which yields u s = b p x − m = u b . the optimality of the corresponding singular arc can be checked with the generalized legendre-clebsch condition of first order, which yields − ∂ ∂u transitioning from an interior arc to a singular arc and vice versa at time τ ∈ [ , t f ] violates the continuity conditions if the condition above holds for any junction time τ , then μ( t , τ ) > for the boundary arc and μ( τ , τ ) ≤ for the singular arc would have to hold simultaneously. thus, a singular arc will not be part of the optimal solution. finally, since h (x (t −)) = and h (x (t −)) = if t = t , ˙ x (t )(u = ) < and the optimal control ( ) ends with a zero segment for t ∈ [ t , t f ], which also satisfies the transversality condition ( ) . if u b (x (t )) ≤ u max does not hold, by analogous derivations to the proof above, it can be shown that adding a finite number n of sequences of a zero arc followed by an u max -arc, until a switch to the boundary arc at time t n + becomes possible with u b (x (t n + )) = u max , fulfills the necessary optimality conditions. for simplicity, this case is excluded in the following elaborations. as a consequence of the above proposition, solutions of problem will exhibit the structure of the generalized bangboundary control policy ( ) . note that the specific optimal policy strongly depends on the choice of w as well as the model parameters. what remains to be determined are the optimal switching times. instead of directly optimizing the switching times t , t , t , t , introduce the arc durations ξ i = t i − t i − , assuming t = t f . thus, the induced optimization problem reads ( ) , ( ) , ( ) , ( ) , ( ) this optimization problem can be simplified further. if w = , i.e., the goal is to minimize only the total number of infections, the op- ( ) is violated. for other values of w , the optimal control is given by ( ) , which denotes two possible policies -one containing a boundary arc, and one without a boundary arc. note that ( ) is explicitly solved on the boundary arc, as already used in the proof of proposition . thus, solving ( ) can be replaced by solving the three aforementioned boundary value problems and choosing the policy that yields the minimal cost. corresponding derivations for the seir model can be found in the appendix. the induced optimization problem ( ) can be recomputed in a data-driven manner to obtain an improved quarantine action, as shown in the following. while a fast response upon an epidemic outbreak is essential, for a newly emerged disease, like in the case of covid- at the beginning of , disease characteristics are highly uncertain. as more data and insights become available over time, it can be expected that the parameter estimates of the models can be improved continuously. this can be achieved, e.g., by minimizing the mean square error loss function between measured state data ˜ ( ) . analogously, this can be done for the seir model with p = { b, m, c} . at any time τ , using the parameter estimates p τ obtained by ( ) and the measured state ˜ x (τ ) , the induced optimization problem ( ) can be re-solved with x = ˜ x (τ ) . assume that the parameter estimates are updated whenever new data becomes available. based on that, a data-driven receding horizon approach is adopted, where the control is obtained by re-computing ( ) , only when the current parameter estimates deviate from the parameter estimates used in the previous computation step evaluated by a function ζ by a pre-defined threshold δ. the similarity function ζ was assumed to be a simple absolute deviation in this work, but it can contain more complex considerations like, e.g., the covariance of the estimates. this leads to algorithm . note that, in general, receding horizon control approaches with uncertain parameters cannot guarantee that hard constraints will not be violated. thus, to increase satisfaction probability, the capacity constraint ( ) can be tightened conservatively based on the confidence of the current parameter estimate p t . the proposed method is applied to data from the sars-cov- outbreak in germany until june . two cases are considered -w = and w = . , which correspond to minimizing only the overall number of isolated individuals and a trade-off between the overall number of isolated individuals and the total size of the outbreak, respectively. the optimization problems are solved with scipy's bvp and minimize routines. most parameters are taken from a report of the german robert koch institute [ ] -a susceptible population of x ( ) = . and an initial number of infections x ( ) = , corresponding to the number of registered infections in germany on march , . the initial numbers of recovered and quarantined individuals are both . for the seir model, an initial exposed individuals number of is assumed. further, a basic reproduction number r t = . is assumed in the uncontrolled models, as well as an inverse mean infectious period in days of m = . . the percentage of infected individuals admitted to an intensive care unit is . % and the mortality is . %. the overall number of intensive care units in germany was α c = . by the end of march [ ] . the assumed maximum control value u max is defined such that it leads to an effective reproduction number ˜ r t = . , i.e., with eq. ( ) given by u max = . . for the receding horizon control approach, publicly available data from rki from march until june , to re-estimate the parameters p = { b, c, m } for this time period, and assume the actual parameters r t = . , c = . and u max = . thereafter. the control is re-computed according to algorithm . figs. and show the model state trajectories, the capacity constraint and the control over time for the uncontrolled (left) and the optimally controlled (right) with the aforementioned parameters for the sir and seir model, respectively. it is notable that for both cases, i.e., for w = and w = . , respectively, the optimal solution is given by a bang-boundary control, when using the sir or the seir model. applying the optimal control to the sir model under the given assumptions, the pandemic is predicted to lead to an expected total number of cases of , and a total number of deaths of . the simulations also indicate that the turnaround (final peak of active cases) should occur weeks after the start of the outbreak. the overall outbreak should be completely overcome after weeks, without a second wave of infections. this closely resembles predictions made in an der heiden and buchholz [ ] and [ ] . fig. shows the result with the data-driven receding horizon control. due to the high estimate of the reproductive number in the initial phase, the maximal isolation control starts earlier, and, overall, applies more isolation due to the parameter uncertainty. note that the control maintains the capacity constraint. in practice, a toggling between bang-bang controls could be prevented by introducing a hysteresis, when a new control policy is computed in algorithm . applying the optimal control under the given assumptions, the pandemic is predicted to lead to an expected total number of cases of , and a total number of deaths of . note that the lower number of infections is a consequence of the longer period of applied quarantine control compared to the preceding simulation example. another consequence of the longer quarantine is a slightly delayed turnaround that should occur weeks after the start of the outbreak. the overall outbreak should be completely overcome after weeks, without a second wave of infections, and while control and capacity constraints are maintained at all times. the optimal quarantine control of an infectious spread was studied assuming an sir epidemic model with permanent immunity and no vaccine availability. the control strategy, which min- parameters : initial parameters p , initial times t , , t , , t , , threshold δ, final time t f output : optimal switching times t , t , t : if p t− = ∅ then : ( ) with data x | [ ,τ ] and initial parameter value p t− . : if ζ (p t− , p t ) > δ then : t , t , t ← solve ( ) initialized with previous t , t , t : p t− ← p t . : end if : return t , t , t imizes the final outbreak state assuming that only limited quarantine control is available and capacity constraints have to be respected, was derived from necessary optimality conditions. the solution is to possibly delay the control action first, and then, if needed, apply maximal isolation. if the capacity boundary is reached, a boundary control maintaining the infection numbers may be applied afterwards, until the infection numbers begin to drop. this result was used in a data-driven receding horizon control approach to improve actions as more data becomes available for a previously unknown disease. the application of the proposed schemes to publicly available data from the outbreak of sars-cov- in germany indicate that a sufficiently delayed and controlled release of the lockdown are optimal, and would result in overcoming the outbreak within one year. note that a constant overall population was assumed throughout this work. effects resulting from population exchange could be addressed, e.g., as suggested in xue et al. [ ] , leading to a stochas-tic induced optimization problem. an interesting remaining question is how to determine the maximal isolation control value, as well as how to map the boundary control values to practical isolation actions. a promising approach for addressing both topics could be using machine learning techniques, e.g., building upon ideas from [ ] . another research direction could be to embed the parameter estimation uncertainty into the data-driven optimization to achieve an improved exploration-exploitation trade-off. the author declares that he has no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. and c denote the exposure and infection rates, respectively. the reproduction number remains as defined in ( ) . analogously to the elaborations provided for the sir model, the most important equations are derived as: following an analogous approach, proposition can be proven for the seir model, yielding an identically structured optimal policy. novel corona virus -ncov: early estimation of epidemiological parameters and epidemic predictions. medrxiv estimation of the transmission risk of the -ncov and its implication for public health interventions nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study prediction of criticality in patients with severe covid- infection using three clinical features: a machine learning-based prognostic model with clinical data in wuhan. medrxiv quantifying the effect of quarantine control in covid- infectious spread using machine learning. medrxiv early dynamics of transmission and control of covid- : a mathematical modelling study infectious diseases of humans: dynamics and control an optimal isolation policy for an epidemic optimal isolation policies for deterministic and stochastic epidemics an explicit optimal isolation policy for a deterministic epidemic model optimal control of epidemics with limited resources time-optimal control strategies in sir epidemic models strategic release of lockdowns in a covid infection model. medrxiv optimal policies for control of the novel coronavirus disease (covid- ) outbreak effective containment explains subexponential growth in recent confirmed covid- cases in china a survey of the maximum principles for optimal control problems with state constraints modelling the sars epidemic by a lattice-based montecarlo simulation extension and verification of the seir model on the influenza a(h n ) pandemic in japan optimization methods for solving bang-bang control problems with state constraints and the verification of sufficient conditions a local field of extremals for single input systems with state space constraints modellierung von beispielszenarien der sars-cov- -epidemie in deutschland projecting the spread of covid for germany. medrxiv a data-driven network model for the emerging covid- epidemics in wuhan, toronto and italy robustification as a tool in modeling biochemical reaction networks the author thanks m.c. lüffe for enriching discussions. including quarantine control analogously to ( ) , the subpopulationsâ evolution in the seir model is governed by the following system of coupled nonlinear ordinary differential equations: key: cord- -fgvbrg d authors: ohara, hiroshi; pokhrel, bharat m.; dahal, rajan k.; mishra, shyam k.; kattel, hari p.; shrestha, dharma l.; haneishi, yumiko; sherchand, jeevan b. title: fact-finding survey of nosocomial infection control in hospitals in kathmandu, nepal—a basis for improvement date: - - journal: trop med health doi: . /tmh. - sha: doc_id: cord_uid: fgvbrg d the purpose of this study was to investigate the actual conditions of nosocomial infection control in kathmandu city, nepal as a basis for the possible contribution to its improvement. the survey was conducted at hospitals and the methods included a questionnaire, site visits and interviews. nine hospitals had manuals on nosocomial infection control, and seven had an infection control committee (icc). the number of hospitals that met the required amount of personal protective equipment preparation was as follows: gowns ( ), gloves ( ), surgical masks ( ). six hospitals had carried out in-service training over the past one year, but seven hospitals responded that no staff had been trained. eight hospitals were conducting surveillance based on the results of bacteriological testing. the major problems included inadequate management of icc, insufficient training opportunities for hospital staff, and lack of essential equipment. moreover, increasing bacterial resistance to antibiotics was recognized as a growing issue. in comparison with the results conducted in targeting five governmental hospitals, a steady improvement was observed, but further improvements are needed in terms of the provision of high quality medical care. particularly, dissemination of appropriate manuals, enhancement of basic techniques, and strengthening of the infection control system should be given priority. recently, nosocomial infections have become a global concern recognized as a major patient safety issue. they not only cause a significant burden on patients but also lower the quality of medical care. in addition, prolonged hospitalization due to nosocomial infections increases costs and unnecessary expenses for the hospital [ , ] . in the healthcare setting, particularly in developed countries, various measures including the organization of infection control teams (icts), preparation of manuals, strengthening of surveillance systems, and training of staff have been taken to assure effective control. however, it is only some decades ago that importance was attached to nosocomial infection control and effective measures were employed, even in developed countries [ ] . in developing countries, where the incidence of infectious diseases is high and environmental conditions of healthcare facilities are poor, nosocomial infections may frequently occur, and some studies have reported a high incidence at healthcare facilities in these countries [ ] [ ] [ ] . effective nosocomial infection control is crucial in the healthcare facilities of developing countries, but in actual fact, attention to it is still limited and control measures are not functioning well in many facilities. furthermore, as implementation of control measures seems to be costly and to consume resources, nosocomial infection control is often given a low priority. severe acute respiratory syndrome (sars), which originated in guangdong province, china in november , spread to more than countries. in many hospitals where sars cases were encountered, nosocomial infections also broke out, causing many casualties along with economic havoc [ , ] . it is not overstatement to say that such outbreaks have heightened awareness regarding nosocomial infection control even in developing countries. in more recent years, epidemics of novel influenza have also posed a threat of nosocomial infections [ ] . these facts made many people realize again the importance of strengthening nosocomial infection control at hospitals in developing countries. some of the authors of the present paper have been engaged in technical cooperation for nosocomial infection control with hospitals in developing countries, recognizing the importance of strengthening control measures in order to enhance the quality of medical care. between and , they have contributed to the promotion of nosocomial infection control in vietnam in collaboration with leading hospitals [ , ] . since , in response to the growing concern regarding nosocomial infections, we have focused our efforts in nepal through collaboration with tribhuvan university teaching hospital (tuth) in kathmandu city, where a technical cooperation project by japan international cooperation agency (jica) had been implemented to strengthen the hospital. following studies including those on hospitalacquired diarrheal diseases [ ] and the prevalence of multiple drug-resistant pathogens [ ] , this survey was carried out as a baseline study aiming to contribute to the improvement of nosocomial infection control at tuth and consequently in kathmandu city. the primary purpose of this study was to evaluate nosocomial infection control conditions and to prepare the basic information needed to provide technical guidance. the subjects of this survey are leading hospitals in kathmandu city (five national hospitals, nine private hospitals, and three other hospitals). the national hospitals included three general hospitals (one out of three was a university hospital; i.e. tuth), one pediatric hospital and one obstetric hospital. all the private hospitals were general hospitals, while three other hospitals included one semigovernmental hospital and two non-profit organization hospitals (these three hospitals were general hospitals). the hospitals play a crucial role in medical care in kathmandu city. a questionnaire was developed based on the form used in the previous surveys in vietnam [ ] . the form consisted of the following items: "general information of the hospitals, control system including manual and infection control committees (icc), equipment and facility preparedness, training conditions, surveillance conditions, expectation for international cooperation and current problems. the contents of each item in the questionnaire are shown in table . the questionnaire was distributed to the hospitals in october and filled out by the hospital staff members who were responsible for nosocomial infection control or the director of the hospital. the recovered data were processed using spss ver for windows. in some hospitals, to determine the actual situation of icc, manuals, current problems and awareness level of hospital staff regarding nosocomial infection control, direct observations were conducted along with a brief interview with the hospital staff responsible for nosocomial infection control or hospital di- current problems requested the hospital to describe the current problems. rector in addition to the information obtained by the questionnaire. in , a questionnaire survey was conducted at five national hospitals in kathmandu city [ ] . these five hospitals were included in this study ( ) . the results of the questionnaire were compared with those of this study ( ), including manuals, iccs, in-service training and preparedness of personal protective equipment (ppe). a comparative statistical analysis of the and results was carried out by the fisher's exact method using spss ver for windows. tuth was established in with the assistance of a grant-aid from the japanese government as the first medical school in nepal, followed by the implementation of a technical cooperation project supported by the jica from to (the corresponding author participated as a team leader). the purpose of the project was to strengthen medical and educational services at tuth. during the above period, technical guidance was conducted in the field of hospital management, clinical medicine, nursing management, laboratory management and medical education. however, nosocomial infection control was not included in the project, probably because awareness regarding nosocomial infection control was still poor in those days even in developed countries including japan. currently tuth is playing a leading role in medical care as well as human resource development in nepal as the oldest and one of the most advanced medical schools. in this study, the current situation of nosocomial infection control at tuth was investigated in detail as a basis for further improvement. during the jica project period, technical guidance was provided, not on nosocomial infection control, but on bacteriological testing as a priority subject. in this study, investigation was performed by direct observation and interviews with heads of the departments of clinical microbiology and pharmacology and doctors of internal medicines, focusing on whether bacteriological testing was utilized for implementation of nosocomial infection control, in addition to detailed observation of the hospital and the questionnaire survey. ethical approval was obtained from the institute of medicine, kathmandu, nepal prior to using the questionnaire in the target hospitals. the hospitals responded to most of the questionnaire items, but for some items, a response was obtained from only hospitals. the average number of beds in the surveyed hospitals was as follows: national hospitals; ( - ), private hospitals; ( - ), other hospitals ( - ). the average number of clinical departments was as follows: national hospitals (excluding the two specialized hospitals); . ( - ), private hospitals . ( - ); other hospitals . ( ) ( ) ( ) ( ) ( ) ( ) . tuth, which is one of the national hospitals, had beds and clinical departments. manuals for infection control were used in . % ( / ) of the hospitals (national / , private / , and other hospitals / ). however, most of these manuals were more than five years old and some of their contents were not considered suitable for recent infectious diseases and antibiotic use. the manuals at three hospitals were considered obsolete. two national hospitals had good manuals with up-todate contents. only three hospitals had manuals for novel influenza. an infection control committee (icc) was established in . % ( / ) of the hospitals (fig. ) . however, a regular icc meeting was held in only two hospitals (once a month, and every three months) and the remaining hospitals held meetings when requested. it was noted that the operations of these committees were far from adequate. no hospitals had an infection control team (ict). equipment and facility preparedness: the number of hospitals which met the standard quantity requirements for disinfectants and personal protective fig. . no hospital was equipped with a sufficient quantity of n masks and goggles. eleven and hospitals responded that n masks and goggles were unavailable, respectively. a total of . % ( / ) of hospitals responded that the preparation level for novel influenza was poor or slight. four hospitals responded that they could prepare isolation rooms to deal with novel influenza/ sars, but no hospital was equipped with negative pressure rooms. only one hospital had a plan of zoning formulated according to the risk of infection. training conditions: current training conditions are summarized in table . six hospitals (four national hospitals, one private hospital and one other hospital) were organizing training programs for their staff (in-service training). regarding future plan, five hospitals responded that they planned to conduct inservice training, and eight hospitals responded that they did not have any plans at the present time but hoped to in the future. among all the hospitals, one had already conducted a training program on sars and/or novel influenza and three hospitals intended to conduct training. surveillance conditions: bacteriological testing was regularly performed for nosocomial infection cases at . % ( / ) of the hospitals and . % ( / ) of the hospitals for some cases. surveillance of nosocomial infections according to reports from clinical departments on clinical signs such as fever, respiratory signs, diarrhea, etc. was regularly carried out in . % ( / ) of the hospitals in the survey (fig. ) . seven hospitals had a strong interest in cooperating with foreign hospitals. a particularly strong expectation was observed regarding research support, information supply, ppe provision, and guidance in constructing an effective control system (table ). among the problems observed in the study were weak icc function, few training opportunities among the hospital staff, inadequate use of antibiotics, shortage of infection control staff, shortage of doctors and nurses and their overload in daily medical practice, shortage of fundamental equipment including ppe, inadequate practice of basic tech- comparison of nosocomial infection control conditions between and at five national hospitals showed an improvement trend. particularly, preparation of ppe and disinfectants remarkably improved as shown in figure (p = . and p = . , respectively), categories in which all five hospitals met the standard quantity. in , four out of five hospitals (except for one specialized hospital) were conducting in-service training, while only one hospital was conducting such training in (p = . ). among these four hospitals, manuals were on hand and an icc was established (p = . ). the first icc in nepal was established in at tuth. since then, an icc meeting has been held once a month. a comparatively good infection control manual was prepared and has been revised according to necessity. inservice training has been conducted for most of the staff at tuth. this study showed a good situation regarding equipment preparedness including disinfectant (sufficient amount), ppe (sufficient amount of ordinary masks, disposable gloves and gowns) along with preparation of isolation rooms. however, incomplete observance of basic techniques such as standard precautions, as well as the need to further strengthen the function of icc, have been pointed out as challenges. performance of bacteriological testing was well carried out in the clinical microbiology department of tuth, and the results were passed on to the clinical side through the drug information office. however, the interview suggested that increased bacterial resistance to antibiotics was a growing issue at tuth. appropriate nosocomial infection control is a key strategy in providing high quality medical care, and effective measures are particularly required in developing countries, where the frequency of infectious diseases is high and environmental conditions of hospitals are poor [ , ] . however, nosocomial infection control is generally not given high priority, and awareness among medical practitioners is still low, a situation that jeopardizes health care functions. in this survey in kathmandu city, steady progress was observed in national hospitals in comparison with the results in . it is particularly noteworthy that awareness among staff and the level of training activities increased with an improvement in the preparedness of essential infection control equipment such as ppe and disinfectants. regarding private hospitals and other hospitals, a comparative study was not conducted using this survey, but an improvement in infection control similar to that of the national hospitals is assumed. however, further efforts to strengthen nosocomial infection control at the target hospitals are still considered necessary. the results showed that the majority of hospitals did not have an up-to-date nosocomial infection control manual, that the surveillance system was not established sufficiently, and that preparations against sars and novel influenza were poor. it is crucial to improve these fundamental systems. moreover, special emphasis should be placed on observance of basic techniques (standard precautions) such as hand hygiene, effective use of ppe and appropriate practice of disinfection [ ] [ ] [ ] . enlightenment activities, such as distribution of manuals and teaching materials and the organization of training courses for medical staff, are very useful and effective for the improvement of nosocomial infection control. an increasing number of hospitals have been establishing iccs in recent years, but the management and implementation of activities need further improvement to achieve effective control measures. hereafter, icts also need to be set up in leading hospitals. furthermore, the detailed status of nosocomial infections and their causative agents should be strictly monitored and properly utilized in clinical practice. among the targeted hospitals in this survey, tuth showed comparatively good results. bacteriological testing, supervised by the jica project, was functioning well and contributing to the surveillance of nosocomial infection based on bacteriological examination and reports from clinical departments for suspected nosocomial infection cases. however, our previous study on pathogens associated with nosocomial lower respiratory infections showed a high frequency of gram negative bacilli such as escherichia coli, pseudomonas aerginosa, acinetobactor baumanii, klebsiella pneumoniae, as well as a high multiple drug resistance rate for isolated bacteria. in addition, a high rate of extended stratum beta lactamase (esbl) producing bacteria was observed [ ] . the spread of multi-resistant bacteria reported by many developing countries is considered to be a facilitating factor in nosocomial infection [ ] [ ] [ ] . methallo β lactamase (mbl) producing bacteria, which originated from india, is also suspected to be spreading to nepal [ , ] . these findings suggest the need for more aggressive measures to tackle this global threat. the appropriate use of antibiotics based on accurate bacteriological testing, along with appropriate guidelines, is a worldwide challenge. nepal, fortunately, has not experienced a sars outbreak, and no human case of avian influenza has been reported to date. on the other hand, awareness of nosocomial infection control seems to be lagging behind countries where a sars outbreak did occur as shown in the study [ ] . when a novel influenza becomes an epidemic and human to human infection is common, nosocomial infections may easily occur as seen in the spanish influenza pandemic of - . appropriate nosocomial infection control is also considered useful for novel influenza control. special importance should be placed on setting up a foundation for appropriate nosocomial infection control in daily practice, training medical staff and establishing a control system, before nosocomial infections become a frequent occurrence. nosocomial infection control is crucial in providing high quality medical care. greater efforts should be focused on training medical staff to enhance basic techniques and establish control systems at ordinary times, not waiting until after an outbreak or epidemic. with such a foundation, it will be possible to promptly apply stringent nosocomial infection control in the event of an outbreak of novel influenza, sars or other emerging infectious disease. these measures will contribute to the reduction of unnecessary costs and can improve the financial condition of the hospital. based on the results of this survey, the authors intend to collaborate with nepalese authorities and further contribute to the improvement of nosocomial infection control. currently, our collaborative activities at tuth are related to basic studies on bacterial resistance to antibiotics and the appropriate use of antibiotics. in addition, guidance on the promotion of standard precautions and surveillance systems is currently being prepared. the results of the present survey are expected to provide baseline data for monitoring the progress of the nosocomial infection control situation at tuth as well as that in hospitals in kathmandu. in this survey, only hospitals in kathmandu city were investigated. infection control conditions are improving in these hospitals but further improvement in the software aspect is still needed to assure high quality medical care. in nepal as well as other developing countries, a significant disparity in the conditions of medical care and the health system exists between major cities and rural areas. in the future, the expansion of nosocomial infection control to hospitals in remote areas will be needed along with the implementation of guidance for hospitals in those areas. hospital-acquired infections in belgian 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cephalosporins among klebsiella pneumonia in chhenai new delhi metallo-beta-lactamase (ndm- ): towards a new pandemia? the authors would like to express thanks to the hospitals in kathmandu city for their cooperation during the implementation of this study. this survey was conducted with the support of grants from the national center for global health and medicine, japan. none. key: cord- -el rembl authors: dantés, héctor gómez; manrique-saide, pablo; vazquez-prokopec, gonzalo; morales, fabian correa; siqueira, joão bosco; pimenta, fabiano; coelho, giovanini; bezerra, haroldo title: prevention and control of aedes transmitted infections in the post-pandemic scenario of covid- : challenges and opportunities for the region of the americas date: - - journal: mem inst oswaldo cruz doi: . / - sha: doc_id: cord_uid: el rembl the coronavirus disease of (covid- ) pandemic challenges public health systems around the world. tropical countries will face complex epidemiological scenarios involving the simultaneous transmission of severe acute respiratory syndrome coronavirus (sars-cov- ) with viruses transmitted by aedes aegypti. the occurrence of arboviral diseases with covid- in the latin america and the caribbean (lac) region presents challenges and opportunities for strengthening health services, surveillance and control programs. financing of training, equipment and reconversion of hospital spaces will have a negative effect on already the limited resource directed to the health sector. the strengthening of the diagnostic infrastructure reappears as an opportunity for the national reference laboratories. sharing of epidemiological information for the modeling of epidemiological scenarios allows collaboration between health, academic and scientific institutions. the fear of contagion by covid- is constraining people with arboviral diseases to search for care which can lead to an increase in serious cases and could disrupt the operation of vector-control programs due to the reluctance of residents to open their doors to health personnel. promoting intense community participation along with the incorporation of long lasting innovations in vector control offers new opportunities for control. the covid- pandemic offers challenges and opportunities that must provoke positive behavioral changes and encourage more permanent self-care actions. since the identification of the new coronavirus (severe acute respiratory syndrome coronavirus -sars-cov- ) and associated disease (coronavirus disease of -covid- ) in january in china, the virus has spread to all continents and the world health organization (who) has reported more than . million infections and . thousand deaths (as of may ). ( , ) unprecedented measures to contain the pandemic and the response of the health systems are being implemented by the countries with a primary focus at the national level. at the local level, hand washing, respiratory hygiene recommendations, and physical distancing are key practices for disease containment, mitigation and suppression. the closing of the borders and social events in urban spaces have also contributed to mitigate the transmission, ( ) although its implementation has been very heterogeneous in each of the affected countries. there is discrepancy between predictive models of covid- behavior; some estimate a significant decrease in transmission between june and september, and on the contrary, additional transmission waves are also forecasted for the second semester of . ( , ) the high occurrence of covid- in tropical cities of latin america ( ) reflects the heterogeneous response of political leaders and the national capacity of public health systems in those countries. the transitions will thus vary accordingly from country to country. ( ) the unexpected expenditures disbursed to implement the prevention and control measures of covid- has put countries in a crisis that will affect not only economic activities but also the regular financing of routine preventive and control programs. we identify that this will be an additional challenge for the health systems and the economy of the countries of latin america and the caribbean (lac). contrary to what will happen in temperate countries, a large population contingent from tropical countries will experience complex epidemiological scenarios that will involve the simultaneous or intercalated transmission of sars-cov- with viruses of dengue, zika and chikungunya transmitted by aedes aegypti (atds). ( , ) another notable aspect is the potential exhaustion of the surveillance systems to investigate and report arboviral cases in a timely manner. in march , the pan american health organization (paho) issued an alert calling attention to the trend of increasing dengue cases in at least countries of the region compared to . ( ) dengue data obtained from health information platform for the americas (plisa) show that weekly dengue cases during were reported on a higher rate than the weekly average of dengue cases reported during the - period, and a sharp decrease in notification of confirmed dengue cases started along with the report of covid- cases in the lac region ( ) (figure) . the decrease in notification was particularly remarkable for south and central america. despite having limited information on the direct impact on human health of the interaction of arboviral diseases with covid- , ( ) it is essential that all efforts be made to protect populations at risk ( ) since aedes-transmitted diseases (atds) mainly affect vulnerable populations living in poor urban or rural areas and in houses with limited access to sewerage and drinking water services. ( , ) the indirect impact of covid- on arboviral diseases may be even greater than the direct one. health seeking behavior has been dramatically modified by covid- , driven by fear of contagion in the population, but also by messages from the health care authorities who recommend staying at home until severe symptoms (breathing problems) develop. in contrast, dengue cases are encouraged to present to health care units for close and early clinical monitoring (i.e., daily hematocrit). clinical management and rapid diagnosis of both diseases in the context of appropriate health facility triage and case management must be developed. for example, maintaining separate care facilities when possible for co-vid- and arboviral diseases is highly recommended. on the other hand, the fear of covid- contagion could also disrupt the operation of vector-control programs. residents may be reluctant to open their doors to health personnel and health brigades may not want to visit sars-cov- high risk areas because of lack of personal protection equipment and potential exposure to the virus. the evaluation of peridomestic settings arises as an alternative by vector control managers but avoiding the interaction with the community. challenges and opportunities -the simultaneous occurrence of aedes-transmitted diseases and covid- in the lac region presents us with very important challenges but also offers opportunities for strengthening health services, surveillance and control of epidemics (table) . the main challenge or obstacle, without a doubt, will be the economic crisis of the countries along with the reorganization of health services disrupted by the health emergency that diverted attention to the diagnosis and care of patients with severe respiratory manifestations. the reorganization of health services and the conversion of hospital spaces to contain the burden of disease caused by covid- has been detrimental to the care of other problems that are equally relevant and demanding in terms of the daily demand for specialized medical services. a similar experience occurred with reproductive, maternal, and child health services during the recent zika epidemic in the americas, and it may well happen again in the future with the resurgence of covid- or any other epidemic of global proportions. lessons learned from these experiences should help us prepare for future health contingencies. particular attention should be put in sustainability of interventions along with gender issues regarding risk of transmission, health care options and control responsibilities in the domestic and urban settings. ( ) financing of training, equipment and reconversion of hospital spaces will have a negative effect on already the limited resources directed to the health sector at the continental level. the indirect impact on health in general will be a collateral cost that will have to be evaluated in due course. there will be countries in the americas that can face this challenge better than others but the countries of the region have been hit by other epidemics without managing to recover financially from recurring health crises: cholera, h n influenza, zika, chikungunya or dengue. for the epidemiological surveillance systems there are challenges and opportunities given the possible seasonal coincidence of transmission, since the triggering signs and symptoms of seeking medical attention in the covid- charts, are fever, general malaise and respiratory discomfort also frequent in arboviruses. the high occurrence of covid- in tropical cities (e.g., iquitos, peru) indicates that temperature may not be as limiting for transmission as some hypothesize, however, the differential diagnosis of dengue, zika and chikungunya will need to incorporate covid- as a diagnostic possibility, although its confirmation triggers very different control actions. the strengthening of the diagnostic infrastructure reappears as an opportunity to prepare the capacity of the regional, nation and state reference laboratories to diagnose a very wide spectrum of infections agents for which the technical inputs, equipment or trained personnel are not available. thus, interaction between academic and public health institutions is mandatory as the pandemic has demonstrated. similarly, proliferation and real-time virtual epidemiological information platforms developed to monitor the pandemic must be available to monitor arboviral diseases and other infectious diseases with the same intensity and frequency with which they have been occurring in mapping the progress of the covid- pandemic. the effort to share and integrate epidemiological information sources for the construction and modeling of epidemiological scenarios becomes an imperative for regional health that allows collaboration between health entities with academic and scientific institutions. examples of such synergy include timely situational analysis, the generation of adequate risk scenarios for the design and selection of more effective control interventions as well as the use of information for education and communication campaigns with the population. an area of opportunity would be the incorporation of communication technologies (tics) in the test and tracing of contacts as well as mobility surveys in support of the early warning surveillance systems. traditionally, the countries of the american region based their control activities with the involvement of the community for the removal and control of domestic larval habitats the development of protective kits or tools so family members can perform the application of insecticide by themselves (spatial repellents, spray bombs) and the free distribution or purchase by the family of appropriate consumer products like spatial repellents, insecticide treated materials, or screens on doors and windows are interventions that should be encouraged and tested on a wider scale. given the new circumstances, home visits could be interrupted by physical distancing and it is essential to start incorporating new control strategies that do not depend so much on home visits by health personnel and are better supported by the promotion of domiciliary interventions that may be developed inside the house by the family nucleus. an additional opportunity is the more efficient use of resources that is based on risk stratification targeting areas in urban settings that are responsible for more than % of historical cases. ( ) in the case of aedes aegypti surveillance and control, the possibility is opened for promoting the incorporation of innovations that do not require and/or can reduce the constant presence of health personnel. ( ) for vector surveillance, the use of rapid larval/pupal surveys or the use of ovitraps in the peridomestic environment with the support of the community could be implemented to improve monitoring of vector densities while the proactive use of targeted indoor residual spraying (tirs) ( ) and contact tracing, ( ) for example, would favor a more extensive and long-lasting control. this paradigm shift becomes imperative and countries in the region have already drawn up action plans for the concurrence of co-vid- and atds. ( , ) of course, the nature of atds will also require action in response to outbreaks. the responsive actions are well-known in each country and must be carried out in a timely manner and have the human, consumables, and financial resources for it. recommendations established in the operational guides must be followed. chemical control (larvicides and adulticides) should be properly applied and selected based on evidence of susceptibility of the local vector populations to guarantee its effectiveness. in the field of risk communication, the covid- pandemic leaves us with lessons, challenges and opportunities that must lead to better information campaigns. risk communication strategies that increase positive behavioral changes that combat disinformation and encourage people to incorporate permanent self-care actions and not only in crisis situations. at the same time, the risks of transmission of covid- and its dispersion throughout the territory limit the full development of the activities that require the action of health agents, but it also creates a great opportunity to promote effective participation of population with the incorporation of protective habits of prevention and maintenance of the domestic environment free of risk factors for the presence of vectors. vector control personnel must be considered essential, and their activities must continue to support the actions required for the effective prevention and control of vtds, even within the contingency imposed by covid- . given the possible scenario of simultaneous transmission of the atds and covid- agents, it is important that the planning of the actions be integrated, combined with the effective multisectoral and population participation where the public and private sectors, schools, the media, tune into the common strategy to deal with health problems. recently, the countries of the region of the americas, with the support of the pan american health organization (paho), unanimously approved the plan of action on entomology and vector control - , ( ) whose objective is to strengthen regional and national capacity for prevention and control of key vectors, and reduce the spread of vector borne diseases (vbd). this plan of action is based on solid technical foundations that seek to strengthen inter-programmatic action, mobilize governments and communities, the improvement of entomological surveillance, the incorporation of new technologies, and the training, creation, and expansion the workforce. given the emergence of covid- , it is essential that countries commit to supporting this plan of action and strengthen their surveillance and control programs in an integrated way where new epidemiological circumstances are seen as challenges and opportunities and much less as disbeliefs or limitations. opportunities to improve control of atds will depend on how well we respond to the covid- crisis and even though the pandemic is still in progress, the challenges ahead demand resources and new capabilities not only to cope with covid- but to strengthen the overall capacity of the health system to respond to any new sanitary crisis. novel coronavirus ( -ncov) situation report - ; coronavirus disease (covid- ) situation report - impact of international travel and border control measures on the global spread of the novel coronavirus outbreak estimation of the probability of reinfection with covid- by the susceptible-exposed-infectious-removedundetectable-susceptible model projecting the transmission dynamics of sars-cov- through the postpandemic period situación de covid- en la región de las américas covid in latin america covid- and dengue fever: a dangerous combination for the health system in brazil covid- and dengue, co-epidemics in ecuador and other countries in latin america: pushing strained health care systems over the edge paho/who -pan american health organization/world health organization. epidemiological update: dengue american health organization. health information platform for the americas (plisa, paho/who), accessed on to alert coinfection of covid- and dengue virus in developing countries in the dengue-endemic area covid- and dengue, co-epidemics in ecuador and other countries in latin america: pushing strained health care systems over the edge the neglected tropical diseases of latin america and the caribbean: a review of disease burden and distribution and a roadmap for control and elimination economic impact of dengue illness in the americas gender mainstreaming as a pathway for sustainable arbovirus control in latin america technical document for the implementation of interventions based on generic operational scenarios for aedes aegypti control evaluación de las estrategias innovadoras para el control de aedes aegypti: desafíos para su introducción y evaluación del impacto manual para aplicar rociado residual intradomiciliario en zonas urbanas para el control de aedes aegypti combining contact tracing with targeted indoor residual spraying significantly reduces dengue transmission efficacy of novel indoor residual spraying methods targeting pyrethroid-resistant aedes aegypti within experimental houses estrategia de control de vectores en el escenario de transmisión simultánea dengue y covid- en méxico plan de acción sobre entomologia y control de vectores - [internet]. in: .º consejo directivo de la ops, .ª sesión del comité regional de la oms para las américas. del al de septiembre del key: cord- - olapsmv authors: xu, zhijie; ye, yuanqu; wang, yang; qian, yi; pan, jianjiang; lu, yiting; fang, lizheng title: primary care practitioners’ barriers to and experience of covid- epidemic control in china: a qualitative study date: - - journal: j gen intern med doi: . /s - - - sha: doc_id: cord_uid: olapsmv background: the coronavirus disease (covid- ) emerged in december and posed numerous challenges to china’s health system. almost million primary care practitioners (pcps) participated in controlling the outbreak. however, pcps’ barriers to and experience of the epidemic control remain unknown and are essential for improving countermeasures. objective: to better understand the barriers pcps faced in covid- epidemic control and their psychological and occupational impacts, and explore potential solutions. design: this qualitative study was conducted through semi-structured, in-depth interviews from february , to march , . participants: a purposive sample of frontline pcps affiliated with either community health centers or township health centers in four provinces of china were recruited. approach: interviews were conducted by telephone, and then recorded, transcribed, and content analyzed. themes surrounding pcps’ barriers to covid- epidemic control, their experience, and potential solutions were iteratively identified using the constant comparative method. key results: of the pcps interviewed, ( %) were women and ( %) worked in rural areas. barriers to epidemic control in primary care included inappropriate pcp scheduling and role ambiguity, difficult tasks and inadequate capacities, and inexperienced community workers and insufficient cooperation. some pcps perceived respect and a sense of accomplishment and were preoccupied with the outbreak, while others were frustrated by fatigue and psychological distress. pcps reported potential solutions for improving countermeasures, such as improving management, optimizing workflows, providing additional support, facilitating cooperation, and strengthening the primary care system. conclusions: due to their roles in controlling the covid- epidemic, pcps in china faced a series of barriers that affected them physically and mentally. support for pcps should help them to overcome these barriers and work efficiently. the current findings provide insight into the challenges and potential solutions for strengthening the preparedness and response of china’s primary care system in future disease outbreaks. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. i n december , a novel coronavirus was detected from a series cases of pneumonia of unknown cause in wuhan, china, which was subsequently named the coronavirus disease (covid- ) by the world health organization (who). [ ] [ ] [ ] as of june , , , cases including deaths had been confirmed in china, and a global pandemic had emerged. in addition, a total of countries had reported over , , confirmed cases and , deaths, and the numbers continue to grow. [ ] [ ] [ ] primary care practitioners (pcps) are essential in confronting the pandemic. for example, almost million pcps in china participated in covid- epidemic control. they worked in collaboration with the community workers and community police in a "joint defense team" led by the neighborhood committee. pcps were responsible for screening suspected cases, visiting residents in quarantine, contact tracing and monitoring, and surveillance at checkpoints, while community workers and community police provide nonmedical support to residents in quarantine. pcps regularly recorded work-related information on forms and uploaded the forms to the neighborhood committee and health administrative departments. similar procedures were undertaken by pcps in singapore. numerous studies have focused on barriers to epidemic control in primary care. a systematic review identified challenges faced by pcps in different countries during previous pandemic response, such as a shortage of personal protective equipment (ppe), limitations of provided information, and insufficient training. a qualitative study showed that pcps experienced difficulties in translating pandemic guidelines into practice. however, the barriers pcps encountered in covid- epidemic control and their solutions have not been explored. another research focus has been the impact of epidemic control on pcps and their experiences. a recent survey showed that general practitioners (gps) in shanghai displayed psychological health problems of varying severity during epidemic control, and . % of gps felt stressed. similarly, previous studies demonstrated that the pandemic outbreak changed pcps' work environment and lifestyle, and led to a series of negative emotions, such as depression, anxiety, and fear. they could also experience symptoms of acute stress disorders or post-traumatic stress disorder (e.g., intrusion, avoidance, and hyperarousal) after the outbreak. therefore, pcps' experience in covid- epidemic control deserves closer attention to inform improvement efforts. to understand pcps' perceived barriers to and experience of performing their tasks in epidemic control, we recruited frontline pcps in china and conducted in-depth interviews using a qualitative design. we aim to understand pcps' perspectives on their work and explore the strategies for improving countermeasures in primary care. from february to march , , we conducted a descriptive qualitative study involving semi-structured, in-depth interviews with purposive samples of pcps. interviews were conducted by telephone because of the nationwide traffic restriction, and they lasted a mean of minutes (range: - minutes). all participants provided verbal informed consent before the interviews began and were not compensated for their participation. the study was approved by the sir run run shaw hospital ethics committee and adhered to the declaration of helsinki. we used wechat, an instant messaging app, to invite pcps to participate in the interviews, using the principle of maximum variation. three family physicians refused to participate because they were not responsible for tasks in epidemic control. participants were affiliated with local government-owned community health centers in urban areas (zhejiang and guangdong province) or township health centers in rural areas (shaanxi and hunan province). participants knew the investigators prior to the interview, but none had worked with the investigators. the sample size was determined using thematic saturation: two investigators (z.x. and y.y.) analyzed the transcripts and notes for newly emergent themes after the first in-depth interviews, and after every or thereafter. we stopped scheduling interviews when additional interview data created little or no change to the codebook and no new patterns or themes emerged. , repeat interviews were not carried out. the interview guide was adapted from relevant qualitative studies involving healthcare workers in infectious disease outbreak, , and was refined through pilot interviews with three pcps to improve appropriateness and clarity (eappendix ). each interview began with a question about the types of tasks participants had performed in epidemic control. probing questions were then used to encourage participants to describe tasks in which they felt their performance was deficient and whether they encountered any barriers to task performance (e.g., how did the barriers or difficulties affect your work?). probing questions also elicited details of pcps' experiences and the occupational and psychological effects of epidemic control (e.g., have you experienced any positive or negative emotion?). at the end of the interviews, investigators encouraged pcps to talk freely about their perspectives regarding strategies that could contribute to improved control measures in primary care. information regarding participants' characteristics was collected before the interviews, which were independently audio recorded and transcribed verbatim by two male general practitioners as interviewers (z.x. and y.y.) who had received training on qualitative interviewing. the interviewers made field notes during the interview when necessary. they independently identified major themes and subthemes via thematic content analysis and developed a preliminary codebook for data analysis based on the first three transcripts. they reviewed transcripts continuously using the constant comparative method to expand existing themes and identify new ideas or themes. the codebook was iteratively refined and finalized via internal consensus until % agreement was reached. maxqda (version . . ) was used in the data analysis and retrieval. transcripts were not returned to participants for comment or correction, but we randomly selected three participants and sent them our main findings via e-mail. they agreed with the themes without modification. we recruited eligible pcps ( family practitioners, internists, surgeons, and pediatrician) from practices ( community health centers and township health centers). of the participants, ( %) were women, and ( %) participants undertook administrative tasks in their medical practice. the mean age of participants was years (range: - years), and the mean duration of practice was years (range: - years) (eappendix ). inappropriate pcp scheduling and role ambiguity. participants described numerous barriers to epidemic control (table ) . some felt overburdened and assigned to unsuitable positions. one family physician explained this feeling using a surprising example: "there were residents [to be quarantined] that day, but the community health center only assigned physicians [to visit them]." others felt confused, as they were asked by the leaders of their community/township health centers to perform low-skilled work and believed they needed an additional supportive workforce. the confusion was described by an internist as follows: "when i came back [from the home visits], i was not free until i disinfected the ambulance. but why not employ a cleaner?" some policies were considered inflexible and pcps felt they limited effective scheduling. for example, the quarantine duration was fixed for everyone leaving the epidemic area, regardless of whether self-quarantine had already been undertaken. one participant commented, "[there is] a one-size-fitsall approach that we need to follow, and it took much more time to address the consequences." these inflexible policies not only increased the unnecessary workload but also may have reduced residents' trust in epidemic control. another reported barrier was excessive inspection and meetings. the government officials and medical experts irregularly visited the community/township health centers and inspected pcps' daily practice of epidemic control, including the material preparation and arrangement, and held meetings to discuss the existing problems and potential solutions with pcps. one participant stated, "it really troubled me that i had to accompany those supervisors, maybe to times a week, and show them what we had done with countless papers and forms and photos." some instructions distributed to pcps by supervisors were perceived as "scratching the surface". in addition, the frequent modification of guidance regarding epidemic control confused pcps. difficult tasks and inadequate capacities. although routine care was largely canceled in many primary care practices, participants frequently noted the deficiencies of the workforce and that they worked for extended hours during epidemic control. pcps were on call hours per day to visit newly quarantined residents. online consultation with residents increased workload during time off. one family physician stated, "i often kept an eye on my mobile phone because the residents often left a message of inquiry in the wechat group waiting for my reply." all participants had limited experience in working during a pandemic and more than half perceived their professional training as inadequate and not tailored to their work of epidemic control in the community. in addition, most institutions lacked ppe (particularly masks and gowns) and pcps generally compromised their safety by reusing ppe. cooperation. pcps in china performed home visits for quarantined residents in cooperation with community workers. a few participants complained that community workers were sometimes inactive in terms of participation in epidemic control: "the home visits should be implemented by a group of family physicians and community workers, but sometimes they arrived late." another barrier pcps encounter was that community workers received inadequate training in epidemic control. as one internist commented, "the temporarily recruited [community workers] had no clinical background; they might fall in a rut and fail to deal with things case by case." participants also described a lack of cooperation between pcps and community workers. one family physician stated, "it might cause a delay if there was any error of communication. sometimes the community workers isolated the resident or days before i received the notice. but [the resident] still needed to stay at home for days." a possible explanation for the miscommunication was that orders were released by different administrative departments with limited previous interaction or experience of cooperation. preoccupation. the heavy tasks and work stress involved in epidemic control resulted in pcps devoting additional energy and effort to their daily practice. one source of pcps' preoccupation with epidemic control was the culture of commitment and sacrifice in the healthcare workforce. one participant expressed his pride in participating in epidemic control: "in my school days, i witnessed the outbreak of sars in and was impressed by the sacrifice of angels in white…so i feel proud to have the opportunity to control the outbreak on the frontline." sense of respect and accomplishment. all participants expressed a sense of respect for epidemic control, which enhanced their relationship with residents. pcps received increased emotional support and appreciation, and their efforts were recognized by residents in the community. participants also expressed satisfaction with the insurance and compensation provided by the government. some pcps felt a sense of accomplishment when the quarantine expired, and the residents they managed were not infected. others were inspired to have greater solidarity with colleagues, described as follows: "it's impressive to see my colleagues bearing the hardship…our cohesion is greater than before and makes it all worthwhile." fatigue. more than half of the participants claimed they experienced fatigue as a result of participating in epidemic control. some participants complained that the work content was beyond their capacity and the requirements were incongruous with their training. the intensive work and tough tasks were described as the main factors affecting fatigue. insomnia was cited as another cause of fatigue. one participant reported that the overwhelming work stress deteriorated his sleep quality, which led to inadequate rest and intensified his experience of fatigue. many other symptoms, such as "memory decline," "weight loss," and "inappetence," were reported as common concomitant manifestations of fatigue. psychological distress. participating in epidemic control made pcps a vulnerable group susceptible to psychological distress ( table ). some experienced fear of being infected, and this fear was intensified by the inadequate supply of ppe and prolonged frontline work. pcps frequently experienced anxiety because they needed to adapt to a fast-paced, highly efficient working environment. some participants felt anxious about errors of omission and residents' complaints. most pcps experienced frustration with the paperwork required for reporting, which was deemed as time-consuming but scarcely conducive to practice. another reason for their frustration was that their efforts and contributions were not always recognized by supervisors. pcps would become angry when residents refused to comply with the quarantine and were offended by scurrilous remarks. however, all participants denied persistent or severe depressive symptoms (e.g., feeling hopeless or suicidal thoughts). most participants found psychological support from their colleagues for their psychological distress, but all participants described a lack of external support, and the reasons were "no available professional psychological support," "too busy to seek for help," and "won't help things at all." "i was constantly taking on new tasks and adapting to new requirement, dealing with things that might come up. i was very anxious at that time." frustration "the guidance was problematic at the early stage of epidemic control, but we had no voice to make a change…i felt helpless and powerless." anger "some villagers were frightened of virus transmission through us physicians and hurled insults at me…i choke down their acrimony…it's very annoying." improving management and supervision. to solve certain problems, such as improper task allocation and inflexible policies, participants generally suggested that administrative departments should develop measures that were more personcentered and based on specific contexts. one participant stated, "i appreciate those officials who listened to our voices, sympathized with our dilemmas, and were capable of providing practical strategies." moreover, streamlining excessive inspection and meetings was suggested by some participants. one surgeon remarked, "…facing a succession of inspec-tions…then i became unmoved. i didn't care about what they asked anymore." optimizing workflow. many participants expressed a wish to work efficiently. for example, paperwork and reporting were frequently mentioned as a barrier to epidemic control and occupied much of pcps' time. strategies involved rational workforce arrangement and internal coordination. as one participant stated, "[the medical institution] could recruit full-time medical assistants to perform the low-skilled tasks, such as the daily statistical report." in addition, participants proposed the option of streamlining the procedures for reporting using an intelligent approach. one physician admitted, "the task of surveillance [at the checkpoints] is getting easier because now we have an identity database to screen the contact history for the travelers." providing necessary support. most participants emphasized an imperative of increasing the supply of ppe to pcps, although they all understood the shortage. one suggested strategy was to "use ppe in a planned way" to ensure the security of the frontline healthcare workforce. other options included "collecting ppe from the public" and "centralized purchasing." some participants thought they lacked the experience of coping with major infectious diseases and needed more professional training. one participant commented, "the online education program helped me gain much knowledge of covid- , but we need lessons more tailored for primary care." facilitating cooperation. participants described the need to reinforce cooperation with community workers. participants noted that it was necessary to identify the division of responsibilities for both sides and strengthen the training and supervision of cooperation. an effective approach would be to establish a mechanism of interaction and communication. as one participant stated, "tacit cooperation cannot be expected in one stroke…if the effect of communication was not significant, then we must try again." strengthening the primary care system. participants unanimously agreed that the covid- outbreak was a challenge to the chinese primary care system. to strengthen this system, participants' recommendations ranged from "increasing investment in primary care institutions" and "developing information technology" to "improving the capacity of healthcare personnel." participants expected a system that was "more resilient", "offered universal coverage down to the community level," and "provided integrated care for residents." primary care is the first line of defense in controlling an epidemic at a community level, but the susceptibility of pcps to tasks and the serious consequences were not fully recognized. to enhance understanding of the current status of covid- epidemic control in primary care, we characterized pcps' perceived barriers and experience. we also examined pcps' perspectives on the solutions that could potentially benefit the primary care system in coping with major infectious diseases. to our knowledge, this was the first qualitative study to explore pcps' work in major infectious disease control in china. the pcps described a series of barriers to epidemic control. aside from the extreme workload, rapidly evolving practice environment, ppe shortage, and inadequate training, which are consistent with international reporting, , participants emphasized specific concerns about inappropriate pcp scheduling and role ambiguity, which complicated their routine work, and insufficient cooperation with community workers, which reduced their work efficiency. these findings highlight new problems within and beyond the primary care system during emergency emergencies. therefore, a feedback channel between pcps and leaders should be established to detect problems in epidemic control. implementation of epidemic control had varied occupational and psychological effects on pcps. some pcps responded to the epidemic proactively because of inner motivation or external pressure, whereas others felt fatigued and expressed psychological distress. evidence suggests that frontline healthcare workers are generally vulnerable to the emotional impact of epidemics. [ ] [ ] [ ] in this study, we inductively identified manifestations of psychological distress among participants-fear, anxiety, frustration, and anger ( table )-most of which were reported as mild in degree and short in duration, and seldomly the cause of absenteeism or disease. our findings provide insights into the factors affecting emotions that primary care managers should acknowledge. for example, pcps felt frustrated with the paperwork of reporting surveillance data not only because it was time-consuming or complex but also it was of little practical value. remarkably, most participants found support from their colleagues, but none received external psychological support, suggesting potential gaps in mental health services for pcps during emergencies. although many studies have reported that positive professional relationships, including dialogue and emotional support, were an essential protective factor for preventing physician burnout, our findings support and expand on the existing knowledge regarding the essential role that the peer support plays in pcps' psychological support during a time of pandemic and workforce scarcity. strategies to help pcps overcome challenges and prevent the primary care system from being overwhelmed are urgently needed. first, health authorities and institutional leaders were expected to provide specific support in terms of material, technology, and mental care to make pcps equipped for epidemic control. it is worth noting that leaders must listen to pcps' concerns and encourage them to ask for help, instead of blaming or criticizing them. an array of feedback channels, such as listening sessions and email suggestion box, could be considered to make pcps' voice be part of the decision-making process. second, the burden of unnecessary work could be reduced to maximize the capacity of pcps during this turbulent time. pcps wished to be freed from non-essential tasks and meetings to perform to their full potential and provide integrated care for residents in the community. it is advisable to consider innovative ways proposed by participants in our study to reduce workload and streamline procedures, such as rational workforce arrangement, effective internal coordination, and establishing an intelligent system of communication and surveillance. third, professional training could be provided to community workers to help facilitate their cooperation with pcps. current healthcare systems in many countries are under extreme pressure, and the use of community workers for the covid response would fill gaps in routine primary care. there is a potential to improve community workers' capacity to deliver a wider range of care for the residents. for example, community workers receiving a basic training program might help pcps manage older people in terms of drug delivery and collection of medical information, and this idea should be investigated in a future study. fourth, steps should be taken to build a more peoplecentered primary care system. several long-standing limitations to china's primary care system, particularly the shortage of professional human resources, substantially increased the difficulty of epidemic control in the community. pcps in china are paid low wages and minimal benefits, receive inadequate training, and experience high rates of occupational burnout, which impede pcps' delivery of integrated and highquality care. therefore, the primary care system should ensure an adequate total income and strengthen the career development opportunities for pcps. there are several limitations in our study. first, the results may not be generalizable to other regions of china because we only interviewed pcps in four provinces. second, pcps other than clinicians (e.g., nurse practitioners) were not included in our study because their scope of responsibilities was narrower than that for clinicians. third, our study was not designed to compare the differences between urban and rural areas. finally, we were unable to triangulate the results with those from other stakeholders, such as policymakers and community workers, but we will consider this in future studies. pcps in china perceived a series of barriers in confronting the covid- epidemic, which had positive and negative effects on their physical and mental health. therefore, effective approaches are urgently needed to help pcps overcome these barriers and work in an orderly and efficient manner. the current findings offer important lessons for policymakers and leaders for improving future control measures. in addition, they highlight the importance of developing the primary care system to strengthen preparedness and response to upcoming health challenges. emerging understandings of -ncov coronavirus infections-more than just the common cold latest data on novel coronavirus who strategic and technical advisory group for infectious hazards. covid- : towards controlling of a pandemic responding to covid- -a 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thematic analysis covid- : gps call for same personal protective equipment as hospital doctors covid- : don't forget the impact on us family physicians supporting the health care workforce during the covid- global epidemic timely mental health care for the novel coronavirus outbreak is urgently needed factors associated with mental health outcomes among health care workers exposed to coronavirus disease occurrence, prevention, and management of the psychological effects of emerging virus outbreaks on healthcare workers: rapid review and metaanalysis online mental health services in china during the covid- outbreak understanding and addressing sources of anxiety among health care professionals during the covid- pandemic national uk programme of community health workers for covid- response early appraisal of china' s huge and complex health-care reforms the primary healthcare system in china acknowledgments: the authors would like to thank frontline primary care practitioners who participated in this study for their timely feedback and contributions to the epidemic control. the authors declare that they do not have a conflict of interest.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons. org/licenses/by/ . /. key: cord- -a ia kxf authors: bellinzoni, r. c.; blackhall, j.; baro, n.; auza, n.; mattion, n.; casaro, a.; la torre, j. l.; scodeller, e. a. title: efficacy of an inactivated oil-adjuvanted rotavirus vaccine in the control of calf diarrhoea in beef herds in argentina date: - - journal: vaccine doi: . / - x( ) - sha: doc_id: cord_uid: a ia kxf abstract we have assessed the potency of an inactivated oil-adjuvanted rotavirus vaccine in beef herds in argentina. two different vaccine trials were conducted. in a small-scale experimental trial, involving pregnant cows ( vaccinated and eight unvaccinated controls), a significant increase in neutralizing antibody titres against different serotypes of bovine rotaviruses was found in both the colostrum and serum of vaccinated cows compared with that of unvaccinated controls. seven days after birth, half of the calves born to vaccinated dams or to control cows were challenged with live virulent virus whereas the other half of both groups were left in contact with the infected calves in order to mimic a natural field challenge. although no statistically significant differences in the rate of protection were observed among the different groups of animals, a larger number of vaccinated calves were protected in comparison with their controls, particularly where animals in contact with infected calves were concerned. secondly, a large-scale field trial was carried out in beef herds involving a total of vaccinated pregnant cows. in farms morbidity and mortality in calves from vaccinated cows were compared with historical data from the previous years at the same locations. in the other six herds, control groups were used to compare data of the same year: cows were vaccinated and were left as controls. taking into account the previous and current incidence of diarrhoea, morbidity and mortality were significantly reduced in of the beef herds tested. vaccine effectiveness was also evident in farms where other enteropathogens such as cryptosporidium and coronaviruses were present, together with rotavirus. diarrhoea in newborn calves is a syndrome in which several causative agents (acting separately or associated in different ways) may be involved. surveys carried out in different countries have shown that rotaviruses, coronaviruses, enterotoxigenic escherichia coli, salmonella spp. and cryptosporidium are the pathogens chiefly responsible for outbreaks of calf diarrhoea - . the control of diarrhoea caused by different pathogenic microorganisms should be possible by the development of preventive strategies through the use of appropriate vaccines , in addition to the general measures currently used, such as management, nutrition and chemotherapeutics. to be effective, these vaccines must be designed rationally and based on the impact that the different enteropathogens have in a definite geographical region; therefore single agents or appropriate combinations of them should be components of these vaccines. other relevant factors in achieving effective vaccination are the type of vaccine (live attenuated or inactivated virus) the formulation, and the route and timing of immunization(s). calves can be protected against rotavirus and enterotoxigenic e. coil by passive immunization, taking advantage of the lactogenic immunity stimulated by maternal vaccination or directly by feeding calves with hyperimmune colostrum obtained from vaccinated dams ' . in contrast, attempts to stimulate active immunity in newborn calves by oral vaccination with live attenuated virus have been unsuccessful so far ' . with regard to the formulation of the vaccine, the most relevant factor is which type of adjuvant to use. when the target for vaccination is the pregnant cow, it has been demonstrated that oil-based adjuvants are more effective than alhydrogel to enhance colostrum and milk antibody titres, at least against e. coli, rotavirus and coronavirus °, . in argentina, neonatal diarrhoea is responsible for important economic losses in beef and dairy herds; morbidity can reach - % whereas mortality ranges between % and %. rotavirus is the major agent associated with diarrhoeal problems in argentinian beef herds . for that reason, after several years of epidemiological studies, it was decided to develop and test an inactivated oil-adjuvanted vaccine with the aim of controlling diarrhoea in beef and dairy herds in argentina. we now report the results of two trials performed with the experimental vaccine. the first trial was a challenge experiment performed in confinement and involving cows; the second was an open field trial involving vaccinated cows in different beef herds. the results showed that the oil-adjuvanted rotavirus vaccine tested was effective in the control of calf neonatal diarrhoea in argentina. the following rotavirus strains were used throughout this work: strain t . this was isolated from a diarrhoeic calf in argentina ( ) and was adapted to grow in ma- cells; it was cloned four times by limited dilution after the sixth passage. according to its antigenic characteristics this strain was classified as serotype (see table ). strain t . this strain, isolated from a diarrhoeic calf in argentina (in ), was adapted to grow in ma- cells and was cloned three times. it was selected for this work because it is one of the few local strains that does not cross-react with the bovine prototype strain, uk. strain uk. this was kindly provided by dr d.r. snodgrass (moredum research institute, edinburgh, scotland, uk). it was passaged times in ma- cells, cloned three times and classified as serotype (ref. ) . strain b . this bovine strain, isolated in iowa, usa and kindly provided by dr g. woode (texas, usa), was passaged nine times in ma- cells and cloned three times. like the local strain t , this strain did not cross react in neutralization assays with the ncdv prototype strain ~ . hyperimmune antisera were prepared in seronegative guinea pigs by injecting intramuscularly #g of the corresponding purified virus emulsified with an equal volume of freund's complete adjuvant. two more injections were given subsequently with the same quantity of virus but mixed with freund's incomplete adjuvant. sera were collected one week after the last inoculation and monitored by elisa and neutralization assays. the assays were carried out as described by gerna et al. ~ briefly, pl virus, diluted with medium to give focus-forming units (f.f.u.) per well, were mixed with # twofold serial dilutions of the antisera to be tested. mixtures were incubated for min at °c and inoculated on top of confluent monolayers of ma- cells growing in -well microtitre plates. the plates were incubated for - h. after fixation with acetone, cells were stained by the ipa technique . the neutralizing titre of each serum was expressed as the reciprocal of the highest dilution that gave % reduction in the number of the stained cells compared with control wells. homologous control viruses were included in the test for each serum. the vaccine was produced with the local rotavirus strain, t , the virus was grown in monolayers of ma- cells. after complete cytopathic effect, cellular fluids containing debris and virus were clarified by centrifugation ( for rain), inactivated with . % formalin ( h at °c), and emulsified with an equal volume of oil adjuvant comprising a mixture of % marcol and % montanide (trademarks of seppic, france). the innocuity of each inactivated antigen preparation was tested by observation of any posible cytopathic effect after three serial passages in ma- cell monolayers. thirteen pregnant hereford heifers were vaccinated twice subcutaneously with ml of the oil vaccine and days before expected delivery. eight heifers remained as unvaccinated controls. calves were nursed by their mothers throughout the experiment; on the seventh day of life half of the calves of both groups were injected orally with tcidso of the t strain. the uninoculated calves were kept in close proximity to those inoculated in order to evaluate the possible occurrence of natural transmission of challenge virus, as might happen in the field ('field exposure'). calves were examined daily for days and were considered diarrhoeic if animals excreted loose or watery faeces for at least days . daily faeces samples were examined for rotavirus, coronavirus, cryptosporidium, enterotoxigenic e. coli and salmonella as described by snodgrass et al. a. cows were bled before first and second vaccination, at calving and at days after delivery. colostrum and milk samples were collected at calving and at , and days postcalving. rotavirus-neutralizing antibodies were assayed in each sample, as described previously . neutralizing antibodies against the uk and t strains (serotype ), the b strain and the t local strain, were determined in colostrum (first milking). in addition, neutralizing antibodies against the t strain were determined in the milk on various days post-calving. seventeen beef herds were selected to perform the field trials, the criterion being presentation of calf diarrhoea problems during ~> years before this study. the beef herds selected for the vaccine trial were located in the provinces of buenos aires and c rdoba, comprising a considerable area of the principal breeding region of the country. these herds had previously been part of an extensive epidemiological study (involving a total of herds) performed by our laboratory to determine the aetiology of the high incidence of neonatal diarrhoea in this region (r.c. bellinzoni et al., unpublished results) . in herds the results were compared with historical controls and in the other six herds approximately two-thirds of the cows were left as unvaccinated controls. a total of cows were vaccinated once, as described above, month before the onset of calving. in four of those herds, unvaccinated cows were separated from the vaccinated animals. in the other two herds, both groups were mixed; in this case, the animals in the unvaccinated group were selected randomly. all the cows involved in the experiment were correctly identified and each one was feeding its own calf. as shown in table , in the vaccinated group only two of seven challenged calves, and none of those kept in close contact with them, developed diarrhoea, whereas in the control group, three of four inoculated animals and two of four uninoculated animals developed diarrhoea. virus excretion did not differ in the inoculated calves from both groups (vaccinated and unvaccinated), but differences were observed in close-contact controls ( table ). all rotaviruses isolated from positive faeces showed identical genomic rna patterns to the t strain used for vaccine production and challenge (data not shown). the results shown in table suggest a higher rate of protection in vaccinated animals compared with unvaccinated controls; however, because of the small number of animals involved, no significant differences were noted on statistical analysis of the data. other difficulties with this type of experiment are, first, assessment of the correct dose of virus to be used for the challenge, and second, the furthermore, management of the herds was not disturbed by participation in the trial. the experiment was carefully controlled by trained veterinarians in close cooperation with farm personnel. during weekly visits to the herds, information about diarrhoeic animals was recorded. the trial lasted until each calf was days old. in all cases, diarrhoea was defined as loose or watery faeces, a situation that usually called for treatment of the diseased animals. in some herds, faecal samples were taken from diarrhoeic calves the year before the trial ( ) and during the present trial ( ), and were examined for rotavirus, coronavirus, cryptosporidium, salmonella and etec as described previously . inability to ensure that all the unchallenged animals kept in close proximity to the infected calves received a pathogenic dose of the virus . coronavirus, cryptosporidium or etec were not detected in any of the diarrhoeic samples analysed. aspects of the immunological status of vaccinated animals were also analysed. as shown in figure , compared with controls, vaccinated cows showed significantly higher neutralizing antibody levels against rotavirus in serum, colostrum and milk until at least days after calving. in addition, the titres of colostrum neutralizing antibodies were sufficiently high to neutralize other strains of rotavirus as well. as can be seen in table , colostrum from cows vaccinated with a strain belonging to serotype also showed neutralizing activity at similar titres against heterologous serotypes. table summarizes the results obtained from the large-scale field trial. in the case of herds a-f, morbidity and mortality data obtained from the last years were compared with data obtained during the current trial. no significant differences were detected in the incidence of diarrhoea and mortality in the contemporary control vaccine, vol. , june aln farms a-f, data relating to the incidence of diarrhoea and mortality in calves born from vaccinated cows were compared with data obtained from an unvaccinated control group as well as with data from the last years at the same farms. in two farms (e and f), controls were mixed (m) with vaccinated animals; in the other four farms (a-d), control animals were separated (s) from the vaccinated areas. in farms c_,-q =, data were compared only with the data of the last years at the same farms; bmean of the last years; centeropathogens detected the year before vaccination: r, rotavirus; co, coronavirus; cr, cryptosporidium; as, controls kept apart from vaccinated animals; m, control and vaccinated animals kept in close proximity groups ( . and . % respectively) when compared with the data of the last years ( and . % respectively) suggesting that, in general, the epidemiological situation did not change significantly in those herds. thus (apart from herd f which showed a low previous incidence of diarrhoea and mortality), vaccination of the cows led to an important reduction in the incidence of diarrhoea and mortality ( . and . %, respectively). in four herds (a-d), vaccinated and control animals were kept apart and in the other two (e, f), controls were mixed with vaccinated animals. in one case (see table , herd f) the incidence of diarrhoea and mortality in the control group was lower than that previously recorded. however, in the other herd kept under similar conditions (table , herd e) the values for the control group were similar to the mean of the last years. thus it is not possible from these results, to reach any conclusions about the effect of maintaining vaccinated animals together with, or separated from, non-vaccinated animals. in the remaining herds (table , herds g-p) the incidence of diarrhoea and mortality in calves born to vaccinated animals was compared with the data of the previous years. the results show again that in of those herds (g-o) diarrhoea and mortality in calves from vaccinated cows ( . and . % respectively) were far less than previously recorded in the same herds ( . and . %, respectively). in nine of the herds involved in this study, the presence of other enteropathogens was studied the year before vaccination. in addition to rotaviruses, coronavirus and/or cryptosporidium was detected. in three herds (table : ll, n and p) animals were sampled during the year of the trial and the same pathogens were found (data not shown). in this context, it should be emphasized that, in eight of the nine herds in which other enteropathogens as well as rotavirus were present, the vaccine was also effective in decreasing morbidity and mortality due to diarrhoea. a growing body of evidence ' indicates that in order to guarantee adequate protection of newborn calves against enteropathogens it is necessary to supply them with continuous and sutticient amounts of neutralizing antibodies against the specific agent. this can be achieved by immunization of the dams, before delivery, with inactivated oiladjuvanted vaccines that will induce the production and excretion of rotavirus antibodies in colostrum and postcolostral milk ' . in this study an oil-adjuvanted inactivated vaccine was assayed in two different experiments. the main purpose of the preliminary small-scale trial, which involved pregnant cows and was conducted with the animals confined and under careful veterinary control, was to evaluate the immune response of such cows after vaccination. the efficacy of this vaccine in protecting calves from experimental challenge was also assayed in this trial. in concordance with previous reports . , the results of this experiment show that the level of neutralizing antibodies against homologous and heterologous rotaviruses in sera and colostrum was significantly enhanced on vaccination of the pregnant cows. under the conditions of this trial the incidence of diarrhoea in calves born from vaccinated cows was lower than those born from unvaccinated controls. an interesting observation was the finding that all four uninoculated control calves were actively excreting rotavirus, indicating that the natural exposure used in this experiment was a valid method of challenge. this finding is important because the dose of challenge virus used in a direct inoculation probably would not be representative of the field conditions in which spontaneous infections are continuously occurring. in this regard, difficulties (overwhelming or inadequate doses of virus) in evaluation of the efficacy of immunization by direct inoculation of calves, have been reported , . the results of the small experimental trial showed that the experimental vaccine was able to induce, in the milk and sera of vaccinated cows, an adequate level of neutralizing antibodies against homologous and heterologous rotaviruses; in addition, the results of the challenge were encouraging. it was therefore decided to test the vaccine in a large .field trial in order to evaluate the protection afforded by the vaccine against circulating field rotavirus. in the field trial the efficacy of the vaccine was assessed by two different approaches: by comparing the incidence of diarrhoea and mortality between vaccinated and unvaccinated groups of animals and by comparison of these data with previous values of diarrhoea and mortality (mean of the last years) at the same farms. the use of previous data to compare the results of vaccination can be problematical because of the possibility that the incidence of the various enteropathogens may change from year to year; on the other hand, when data are compared with contemporary controls that are in close contact with the vaccinated group, the incidence of diarrhoea and mortality in these controls may be underestimated because of herd immunity . in order to avoid this problem, control and vaccinated groups can be separated during the trial. the experiment shown in table was designed to test the comparison methods. as judged by the results obtained, it was clear that the vaccine was effective in protecting calves born from vaccinated dams independently of the method used for comparison (historical or actual values of incidence of the disease). it is likely that the higher level of protection against rotavirus infection of calves born to vaccinated dams, compared with those born to control cows, was acquired through colostral immunity. all the herds selected for this work had experienced problems with calf diarrhoea during at least the three years before the trial. nine of the herds studied were sampled the year before the trial and, besides rotavirus, coronavirus and/or cryptosporidium were detected; in three of those herds the same enteropathogens that had been found the year before were detected again at the moment of the trial (see results) , suggesting that there were no significant changes in the epidemiological situation at the trial locations. this observation is relevant because the vaccine was equally effective in herds where rotavirus alone, or where rotavirus and the other two enteropathogens, were present. these results are in contradiction to those reported by snodgrass who found that, in two herds in which rotavirus was associated with cryptosporidium, rotavirus vaccination was effective in diminishing the excretion of rotavirus but not in controlling diarrhoea. the reason(s) for such discrepancy are at present obscure and further work with a larger number of herds and animals is necessary to elucidate this problem. however,judging by the results obtained in this work, it is tempting to speculate that under the particular conditions of cattle breeding in our country, rotaviruses could be the agents that play a major part in determining diarrhoea in newborn calves. in this regard, it is likely that biological, nutritional, environmental or management factors could influence the persistence of a given pathogen in the field, or could well influence the degree of pathogenicity of a given infectious agent or different combinations of agents . the results of this work have confirmed and extended the results of snodgrass and colleagues regarding the effectiveness of an oil-adjuvanted inactivated rotavirus vaccine in controlling (in this case) diarrhoea in beef herds in argentina. these data, obtained from vaccinated cattle in different herds, represent the largest field trial so far for this type of vaccine. pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea infectious agents associated with neonatal calf disease in south western idaho and eastern oregon aetiology of diarrhoea in young calves microbiology of calf diarrhoea in southern britain vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity evaluation of a combined rotavirus and enterotoxigenic escherichia coli vaccine in cattle passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or nonimmunized cows intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody laboratory experiments or viral vaccination of calves against rotavirus or coronavirus induced diarrhoea. zentralbl. veterinaermed passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin g antibody secretion in milk evolution des anticorps anti-rota dans le lait de vaches traitees en fin de gestation soit par le vaccine anti-rota complet, soit par i'adjuvant seul incidence of rotavirus in beef herds in argentina serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization antigenic relationships among some bovine rotaviruses: serum neutralization and cross-protection in gnotobiotic calves serotyping of cell culture adapted subgroup human rotavirus strains by neutralization bovine rotavirus serotypes and their significance for immunization bovine milk immunogtobulins for passive immunity to infantile rotavirus gastroenteritis the authors are indebted to the veterinary surgeons and farmers who participated in this trial and to andros bellinzoni and alvina lorensi for technical assistance. this work was supported by consejo nacional de investigaciones cientificas y t~cnicas (conicet), secretaria de ciencia y trcnica (secyt) and the swedish agency for research cooperation with developing countries (sarec). key: cord- -n ciwy authors: cheung, wing; ip, wan-yim; chan, dominic title: maternal anxiety and feelings of control during labour: a study of chinese first-time pregnant women date: - - journal: midwifery doi: . /j.midw. . . sha: doc_id: cord_uid: n ciwy abstract objective to explore and examine the relationship between maternal anxiety levels and feelings of control during labour among hong kong chinese first-time pregnant women. design an exploratory descriptive correlation design. data were collected on three occasions: during latent phase of labour, during active phase of labour and within – hrs after delivery. setting an obstetric unit of a public teaching hospital in hong kong. participants a convenience sample of hong kong chinese first-time mothers. measurements and findings the labour agentry scale (las) is a self-report scale designed to measure feelings of control during childbirth. a visual analogue scale for anxiety (vas-a) was used to measure women's self-reported level of anxiety during labour. pearson product moment correlation coefficient test indicated a significant negative relationship between the feelings of control and maternal anxiety during labour. no statistical relationships were detected between women's attendance at antenatal classes and feelings of control during labour. key conclusions the study showed a significant negative relationship between maternal anxiety and feelings of control during labour. implications for practice midwives should work with women to enhance their personal control during labour and satisfaction with their birth. the insignificant relationship between attendance at antenatal classes and feelings of control suggests the need to evaluate the content of childbirth education in order to empower women's control during labour. traditionally, maternity services have mainly focused on reducing perinatal and infant mortality rates, whereas women's feelings and experience of the childbirth process have tended to be neglected (martin, ) . in the past decade, growing recognition of the importance of woman-centred maternity care has shifted the emphasis of the importance of care to promote pregnant women's psychosocial health (lavender et al., ) . recent studies have suggested that feelings of control during labour are one of the important factors contributing to maternal childbirth satisfaction (gibbins and thomson, ) , and women should be empowered through knowledge of what to expect from accurate information received about the birth process. as studies exploring the concept of feelings of control during labour and the relationship with maternal physical and psychological labour outcomes are limited, the present study was undertaken to look at these issues in more detail. childbirth is a stressful event, and maternal anxiety is known to be associated with a less positive experience and lower satisfaction with the birth (waldenstrom et al., ) . anxiety was found to rise concomitantly with the level of pain experienced throughout labour in women of all personality types (connolly et al., ) . spiby et al. ( ) stated that a hierarchy of methods, mainly focusing on coping with labour pain, might seriously limit women's psychosocial needs. wuitchik et al. ( ) showed that anxiety could persist even after adequate pain relief was achieved. morgan et al. ( ) concluded that, although analgesia was effective for pain relief, it could provoke anxiety and affect the birth experience. various methods have been suggested to reduce the level of anxiety and labour pain. these include childbirth education (brewin and bradley, ) , nonpharmacological and pharmacological pain-relief methods (moir, ; schuiling and sampselle, ; hodnett, ; lowe, ) , music therapy (browning, ) , support from midwives (sigridur and sigfridur, ) and significant others (tarkka and paunonen, ) . however, the effectiveness of these methods remain inconclusive. lowe ( ) has explained that how women express, manage and cope with labour discomfort is individual and dependent on a variety of factors. recently, gibbins and thomson ( ) pointed out that maternal feelings of control during labour could be the most vital predictor of an enjoyable birth, and may help in decreasing the level of anxiety during pregnancy. the definition of feelings of control in the maternity context was given by hodnett and simmons-tropea ( ) on the basis of the refined work of tiffany et al.'s ( ) model of control. they defined feelings of control as the sense of mastery over internal and environmental forces by personal initiation of choice that was self-actualising, active, responsible, creative and controlling one's own density. recently, green and baston ( ) categorised the senses of control into three dimensions: control of what health-care professionals are doing, control of one's own behaviour and control during a contraction. it seems that this categorisation of control provides a more specific and measurable way for women and caregivers to evaluate control during labour. some investigators have suggested that feelings of control are positively related to the childbirth experience and satisfaction with this (berg and dahlberg, ; cheung, ) . in evaluating the expected and experienced feelings of control of women during labour, hodnett et al. ( ) found no significant difference in experienced control between women having expectant management of pre-labour rupture of their membranes and those who had their labour induced. however, women had significantly higher labour agentry scale (las) scores when they experienced continuous caregiver support (hodnett and osborn, ) , spontaneous birth without continuous fetal heart monitoring (hodnett, ) and decreased usage of pharmacological pain-relief measures (hodnett and abel, ) . moreover, green et al. ( ) noticed that feelings of control during labour enhanced women's emotional well-being after labour, and these positive experiences persisted even though they were different from their expectation. feelings of control have also been linked to various variables related to the childbirth experience. brewin and bradley ( ) found childbirth preparation was positively associated with women's perceptions of personal control over the discomforts experienced during childbirth, such as anxiety and pain. gibbins and thomson ( ) reported that information giving during labour and participation in decision-making were crucial in helping women achieve feelings of control. brown and lumley ( ) and jacoby ( ) found that medical intervention during the labour period was negatively related to overall maternal satisfaction and feelings of being in control. however, other researchers (blanche et al., ; gibbins and thomson, ) claimed that labouring women who received medical intervention still found they experienced a sense of control as long as they were fully informed of events during their labour. previous studies have reported inconsistent findings in chinese women's perceptions towards their feelings of control and participation in decisions about their pregnancy and birth. woollett and dosanjh ( ) indicated that chinese women who lived in east london perceived personal involvement in decision-making as very helpful, and were more committed to seeking childbirth information than non-asian women. in investigating the difference in choice and control as experienced by chinese and scottish childbearing women in scotland, cheung ( ) found that chinese women preferred to give birth without medical intervention and to be well informed during labour. in contrast, a recent study (ip et al., ) revealed that chinese pregnant women had a strong expectation about the level of social support that should be provided by their nurses and partners during labour; however, their expectations of their own responsibilities towards how they coped with childbirth were relatively low. previous studies investigating the relationships between maternal feelings of control and levels of anxiety during labour are limited. in the present study, we investigated the relationship between hong kong chinese first-time pregnant women's anxiety levels and their feelings of control during labour. the relationships among the women's sociodemographic characteristics and physiological variables of labour and their feelings of control were also investigated. it was envisaged that the results of this study could ( ) increase understanding of the psychological parameters of chinese women in childbirth; ( ) help in explaining women's unspoken high anxiety level and its relationship with their feelings of control during labour; and ( ) help midwives to develop appropriate strategies to enhance positive childbirth experience for their clients. an exploratory descriptive correlation design was used to examine the level of maternal anxiety and feelings of control during labour among hong kong chinese first-time pregnant women, and to examine the relationship between level of maternal anxiety and feelings of control reported by the women. a convenience sample of primigravid women in their latent phase of labour was recruited from the labour ward of a public teaching hospital in hong kong. they were chinese, aged years or over, could read and speak cantonese and had not developed complications during pregnancy. of the women recruited, four were excluded from the study as one required emergency caesarean section because of suspected fetal distress, and three required epidural analgesia for pain control, giving a final sample of ( % response rate). according to cohen ( ) , this was the recommended minimal sample size to determine study relationships with a medium effect size (r ¼ . ) at % level of significance and a power of . . the study was carried out in the obstetric unit of a public teaching hospital in hong kong during the period of the severe acute respiratory syndrome (sars) endemic. as a precautionary measure, no visitor was allowed to enter the hospital. the dominant model of care in the unit was medical and supported the active management of labour. this meant that most women had their labour induced or augmented; all women in labour abstained from food and drink, and received continuous electronic fetal monitoring. in addition, each woman was required to have -hourly and -hourly vaginal examinations, respectively, during the latent and active phases of their labour. the use of continuous fetal monitoring restricted women to a lateral position on the labour ward bed, and to give birth in the lithotomy position. moreover, episiotomy was routinely carried out for every primigravida. women at low risk were classified as a midwifery case according to the hospital protocol; this meant that women in labour were cared for by midwives without the involvement of an obstetrician unless a complication was detected. one midwife usually took care of more than one woman in labour at any given time. about hrs after giving birth, women were transferred to a postnatal ward where a new team of midwives took care of them and their babies. the las was developed by hodnett ( ) . it is a self-report scale designed to measure feelings of control during childbirth. the las consists of short affirmative statements (e.g. 'i felt confident' and 'i felt tense'). respondents were asked to rate each statement on a seven-point likert scale from (representing rarely) to (representing almost always). possible total scores for the las range from (indicating feelings of control rarely) to (reflecting feelings of control almost always). the reported internal consistency of the las ranges from . to . (hodnett and abel, ) . hodnett and simmons-tropea ( ) claimed that las scores remained stable at weeks, month and months postpartum. in the present study, the chinese version of las (c-las) was used, which has demonstrated high internal consistency with a cronbach's alpha of . , comparable to the previous findings reported by law ( ) . women's self-reported levels of anxiety during labour were measured with a cm vas, with chinese verbal anchors at either end ranging from (not at all) to (very much so). women indicated their response by adjusting the pointer on the vas-a ruler. the intensity of anxiety was calculated in centimetres by measuring from zero end of the ruler to the woman's mark. the criterion-related coefficients for measuring anxiety in childbirth are . - . (guiffre, ) . a demographic sheet was used to record the woman's socio-demographic data and information on their attendance at antenatal classes. obstetric data collected included mode of vaginal delivery, duration of labour, types of analgesia used and medical intervention. ethical approval was obtained from the ethics committees of the institutions concerned. a pilot study was conducted with five women in the obstetric unit of the target hospital. revisions and refinements were made to the instrument and data collection procedure before the main study. minor corrections were made to the instrument to achieve a better understanding by the women. women who met the inclusion criteria were invited to participate in the study. each woman was provided with a consent form along with an information sheet outlining the purpose of the study, the right to refuse or withdraw at any time and assurance of confidentiality of the collected data. data were collected by the researcher in the labour and postnatal wards between june and december on three occasions: ( ) during the latent phase of labour when cervical dilatation was within - cm with effaced cervix (bennett and brown, ) ; ( ) during the active phase of labour when the cervical dilatation was within - cm (bennett and brown, ) ; and ( ) within - hrs after the delivery. on the first two occasions, women were asked to rate their anxiety levels at the respective state of labour by adjusting the pointer of the vas-a ruler when they were able to respond during the period of uterine relaxation. the last set of data was collected in the postpartum ward where women were asked to recall and rate their overall level of anxiety and feelings of control during labour, using the vas-a ruler and c-las, respectively. women were not asked to rate their anxiety levels during the transitional phase when cervical dilation was within - cm (bennett and brown, ) , as they were likely to be suffering from the most intensive pain during this phase and would have found it difficult to respond to the questions attentively (niven and murphy-black, ) . data were analysed using the statistical programme for quantitative data analysis, spss for window version . . descriptive and inferential statistics were performed on variables. frequencies, means and standard deviations were used to describe the characteristics of samples. pointbiserial formula was used to correlate c-las with dichotomous variables, such as antenatal class attendance, marital status, mode of delivery and obstetric intervention. interval and ratio data, including maternal age, vas-a and c-las scores, were analysed using pearson product moment correlation coefficient. the relationship between c-las and those ranking data, including educational level and family income, were analysed using spearman's rho test. all correlation tests were two-tailed, with a value of po . considered significant (bryman and cramer, ) . three pairwise comparisons were made by using a paired t-test to determine if women differed in their mean anxiety score in the three time slots, with bonferroni correction made to the level of significance of . ( . / ) (munro, ) . data completed on women were included in the analysis. the mean age of women was years (sd ¼ . ), and the mean gestational age of their pregnancy was . weeks (sd ¼ . ). most women were married ( %), % reported to have planned their pregnancy, % had attended antenatal classes, % had completed secondary education and % claimed to have no religious belief. financially, % of women reported that their monthly family incomes were less than hk$ , , lower than the territory's average of hk$ , (census and statistics department of hksar, ). the mean duration of labour was . hrs (sd ¼ . ). most women had normal vaginal delivery ( %). in the area of pain relief, % received pethidine and entonox inhalation, % required augmentation of labour that included aminotomy or oxytocin infusion, and % of women did not require any medical intervention. the mean scores of maternal anxiety reported at the latent phase (anxiety ) and active phase (anxiety ) were . (sd ¼ . ) and . (sd ¼ . ), respectively; the mean score of the recalled overall anxiety during labour (anxiety ) was . (sd ¼ . ). pearson's product moment correlation analysis showed that anxiety was positively and significantly related to anxiety (r ¼ . , po . ) and anxiety (r ¼ . , po . ). furthermore, anxiety was related significantly to anxiety (r ¼ . , po . ). according to the paired t-tests, a significant difference was found between anxiety and anxiety (po . ). another significant difference was also found between anxiety and anxiety (po . ). however, no significant difference was found between anxiety and anxiety (p . ). the c-las scores reported by the women in the postnatal ward ranged from - , with a mean score of . (sd ¼ . ). pearson's product moment correlation analysis revealed a statistically significant negative relationship between feelings of control and maternal anxiety during the latent phase (r ¼ . , po . ), active phase (r ¼ . , po . ) and recalled overall anxiety during labour (r ¼ . , po . ). pearson's product moment correlation analysis revealed no significant relationship between maternal age (p . ) and feelings of control. pointbiserial formula showed no significant relationship between women's feelings of control, planned pregnancy, religious belief, attendance of antenatal class or marital status (p . ). spearman rho analysis revealed no significant relationship between women's feelings of control, educational level and family income (p . ). pearson's product moment correlation revealed no significant relationship between the duration of labour and feelings of control (p . ). pointbiserial formula showed no significant relationship between feelings of control, usage of analgesia, mode of delivery and obstetric intervention (p . ). the convenience sampling method and the small sample size suggest that the findings of the current study cannot be generalised to the total population. maternal feelings of control and anxiety during labour are soft phenomenon that may change over time from the birth as women become 'wrapped up' with their baby (mccrea and wright, ) . this may affect the accuracy of the recalled information. moreover, the vas is used as the selfreport tool and may overestimate or underestimate the anxiety experienced by the women during labour. a replicated study of anxiety and feelings of control with a larger sample size is recommended. as expected, the retrospective measure of the overall anxiety level during labour correlated highly with the measures taken at the time of labour. the insignificant differences between the recalled overall anxiety scores and anxiety scores during the active phase of labour may shed some light on the use of retrospective measures in studies that have difficulties in gaining access to the labour ward. some previous investigators (simkin, ; mccrea and wright, ) have argued that the recall of labour experience might be subject to the 'halo' effect, as this was a recall of negative aspects of childbirth. notwithstanding, redelmeier and kahneman ( ) suggested that it was impossible to retain every aspect of pain episode in memory, and the retention of a memory of pain at its worst may be maximally efficient. this theoretical explanation could be extended to recall anxiety level, and, as such, the postnatal assessed anxiety could represent a measure of anxiety at its most intense. more studies about anxiety of diverse cause are necessary before one can conclude that postnatally assessed anxiety does represent a measure of anxiety at its most intense. hodnett and abel ( ) concluded that the mean las scores of homebirth primigravida ( . , sd ¼ . ) were higher than those who gave birth in hospitals ( . , sd ¼ . ). the mean c-las score reported by the chinese primigravida in the current study was . (sd ¼ . ), which is comparatively lower than the mean las scores reported in hodnett and abel's ( ) study. this suggests that the environment of birth might affect the woman's feelings of control. women who had a home birth indicated that they anticipated greater control over decision-making, greater commitment to non-medicalised childbirth, more freedom of mobility and avoidance of intrusive medical procedures (hodnett and abel, ). in addition, hodnett and abel ( ) reported that those women who gave birth in hospitals were allowed to ambulate until delivery. in contrast, the hong kong chinese women were confined to bed and positioned laterally, with continuous fetal heart monitoring throughout their entire labour. in an earlier study, hodnett ( ) reported that women with continuous fetal heart monitoring reported lower las scores than those without continuous fetal heart monitoring. women claimed that the external electronic monitor interfered with their movement and ability to attain a comfortable position during labour, and they consequently required more analgesia (hodnett, ) . previous randomised-controlled trials have revealed no significant differences in the labour outcome between low-risk women during labour receiving intermittent auscultation and those with continuous electronic fetal heart monitoring (feinstein et al., ) . in this study, one caregiver usually took care of more than one woman during labour at any given time. besides environmental influences, the support of the caregiver is vital to the feelings of control in the women during labour. this was reinforced by hodnett and osborn ( ) who studied labouring women receiving continuous one-to-one caregiver support during labour. hodnett and osborn ( ) reported higher las mean score ( . sd ¼ . ) than the chinese women during labour in the current study. this suggested that there would have been room for improvement in the current midwifery practice in hong kong, with the objectives for balancing economic constraints while providing quality care. heavy reliance on a technological and medicalised approach to birth at the study unit may reflect that the administrative health policies do not value or understand the time intensiveness of being with women, and this may influence women's satisfaction and their postnatal health and well-being. midwives are encouraged to initiate dialogue with obstetricians to look for ways of improving women's birth environment. waldenstrom et al. ( ) emphasised that the sense of control during childbirth came from the opportunity to receive support from significant others or caregivers. gibbins and thomson ( ) added that the perceived partner's support during labour was identified by the women during labour as a crucial factor in helping them maintain control and cope with the challenge of labour. as the current study was conducted during the sars endemic in hong kong, when husbands were not permitted to accompany labour, the influence of husband's support on women's sense of control could not be explored. it would be worthwhile replicating the study with the husbands present during labour in future. nevertheless, numerous studies have suggested that women might gain a greater sense of control by relinquishing decision-making rights to their entrusted caregivers whom they perceive to know and provide the best care (bluff and holloway, ; halldorsdottis and karlsdottir, ; green, ) . miller ( ) went further and asserted that when an individual believed another person possessed a more reliable control response than her own, the individual would prefer not to have any control (miller, ) . midwives were always held in high regard by women during labour in hong kong (holroyd et al., ) . their attitudes and the care provided play an important role in helping women to get through the long, intense and tumultuous childbirth (page, ) . it was apparent that the mean c-las score reported by the hong kong chinese women during labour was comparatively lower than that reported in western countries. the differences in the cultural background among these women in labour should also be taken into consideration. in a study on the perception of choice and control by chinese and scottish childbearing women in scotland, cheung ( ) found that chinese women tended to focus on the safety of their babies and themselves, and they required encouragement to have self-control over their behaviour in coping with labour pain. on the other hand, scottish women were more concerned about what had happened and what was done to them (cheung, ) the findings suggested a negative relationship between the feelings of control and maternal anxiety. in other words, women in labour who perceived better personal control over the labour process reported a reduction in the level of anxiety. this is echoed by mineka and kelly ( ) who reported that the person that preferred to take control found it effective in lowering anticipatory anxiety as well as reducing the effect of aversive events. the insignificant relationship between maternal feelings of control and use of pain relief during labour found in this study was consistent with findings reported by ip et al. ( ) that chinese women have low expectations of their own ability to cope with pain. moreover, results from the current study were also consistent with the findings reported by green and baston ( ) that the duration of labour was not related to feelings of control and that there were no significant relationship between the type of obstetric intervention and article in press the feelings of control. evidently, obstetric procedures may not decrease a woman's feelings of control. stark ( ) reported that a mature primigravida may have greater confidence in her ability to maintain control and cope with labour, as her life experience and knowledge would enable her to face the challenges of childbirth. mercer ( ) asserted that women aged years and older had greater flexibility and personality integration than younger mothers. however, no significant relationship was found between maternal age and feelings of control in the present study. one possible explanation was that most participants were below years and younger mothers under the age of years were excluded from the study. no statistical relationship was detected between participants' attendance to antenatal class and feelings of control. it is contrary to the findings reported by brewin and bradley ( ) that childbirth preparation was positively related to personal perceived control. although most of the women in the present study claimed to have attended antenatal classes ( . %), the number of attendances and the content of information obtained were beyond the control of the study. accordingly, evaluative studies on childbirth education regarding the empowerment of personal control over childbirth are recommended. in this study, we found a significant negative relationship between maternal anxiety and feelings of control during labour. a sense of control is an important predictor to a positive childbirth experience (green et al., ; gibbins and thomson, ; green and baston, ) . childbirth preparation is crucial to enhance women's confidence towards labour and thus her personal control (thompson, ) . the lack of a relationship between women's attendance at antenatal classes and feelings of control suggests the need to evaluate the content of traditional childbirth education to enhance women's control, and inform ways in which midwives could work with women to attain satisfactory childbirth. a phenomenological study of women's experiences of complicated childbirth dysfunctional labour: a randomized trial they know best: women's perception of midwifery care during labour and childbirth perceived control and the experience of childbirth satisfaction with care in labour and birth: a survey of australian women using music during childbirth quantitative data analysis for social scientists. routeledge, london. census and statistics department of hksar choice and control as experienced by chinese and scottish childbearing women in scotland a power premier labour as a psychosomatic condition: a study on the influence of personality on self-reported and pain foetal heart rate auscultation: comparing auscultation to electronic foetal monitoring women's expectations and experiences of childbirth commentary: what is this thing called control? feeling in control during labour: concepts, correlates and consequences expectations, experiences and psychological outcomes of childbirth: a perspective study of women validation of a visual analogue scale for pain and anxiety measurement in childbirth. unpublished doctoral dissertation journeying through labour and delivery: perceptions of women who have given birth patient control during labour. effects of two types of foetal monitors pain and women's satisfaction with the experience of children: a systemic review person-environment interaction as a determinant of labour length variables women's evaluations of induction of labour versus expectant management for prelabour rupture of the membrane at term effects of continuous intrapartum professional support on childbirth outcomes the labour agentry scale: psychometric properties of an instrument measuring control during childbirth hong kong chinese women's perception of support from midwives during labour childbirth expectations of chinese first-time pregnant women women's preferences for and satisfaction with current procedures in childbirth: findings from a national study a prospective study of women's views of factors contributing to a positive birth experience the relationships between self-efficacy of childbirth, feelings of control and perception of pain during childbirth the pain and discomfort of labour and birth the nature of labour pain how do you count maternal satisfaction? a user commissioned survey of maternity services satisfaction in childbirth and perceptions of personal control in pain relief during labour the relationship of developmental variables to maternal behavior controllability and human stress: method, evidence and theory the relationship between anxiety, lack of control and loss of control pain relief in labour effectiveness of pain relief in labour: a survey of mothers statistical methods for health care research memory for labour pain: a review redefining the midwife's role: changes needed in practice patients' memories of painful medical treatment: real time and retrospective evaluations for two minimally invasive procedures comfort in labour and midwifery art journey through labour and delivery: perceptions of women who have given birth just another day in woman' life? part : nature and consistency of women's long-term memories of their first birth experiences selected coping strategies in labour: an investigation of women's experiences psychosocial adjustment during pregnancy: the experience of mature gravidas social support and its impact on mothers' experiences of childbirth will it hurt less if i can control it? a complex answer to a simple question a model of control the childbirth experience: a study of new mothers the ideas and experiences of pregnancy and childbirth of asian and non-asian women in east london relationships between pain, cognitive activity and epidural analgesia during labour key: cord- -tfrjw authors: ledzewicz, urszula; schättler, heinz title: on the role of the objective in the optimization of compartmental models for biomedical therapies date: - - journal: j optim theory appl doi: . /s - - - sha: doc_id: cord_uid: tfrjw we review and discuss results obtained through an application of tools of nonlinear optimal control to biomedical problems. we discuss various aspects of the modeling of the dynamics (such as growth and interaction terms), modeling of treatment (including pharmacometrics of the drugs), and give special attention to the choice of the objective functional to be minimized. indeed, many properties of optimal solutions are predestined by this choice which often is only made casually using some simple ad hoc heuristics. we discuss means to improve this choice by taking into account the underlying biology of the problem. optimal control is a methodology to obtain well thought through solutions to practical problems for dynamical processes. it has found numerous applications in the sciences and has become a staple of engineering design, but applications to biomedical problems are less well known. yet, optimal control as a tool in the analysis of mathematical models of cancer chemotherapy dates back to the s and s (e.g., see [ ] [ ] [ ] [ ] [ ] [ ] ), and these efforts have continued actively throughout the years to the present day (e.g., see [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the many references in [ ] ). in the modeling, considerable attention is given to the formulation of the underlying dynamics. based on the tremendous progress that has been made over the years in understanding biomedical problems, and cancer in particular, even minimally parameterized models are generally well thought through and realistic. as a matter of fact, however, considerably less effort is made when the objective is formulated that will be imposed on the process. omnipresent are the papers in which the formulation of the criterion at best is a mere after-thought rested in questionable heuristics based on the notion of a "systemic cost"-whatever that may be. naturally, the choice of the objective in an optimal control problem is a very important aspect of the overall approach and quite often it is here where what will be eventual properties of the optimal solution are already determined. one should not confuse this with obtaining meaningful information on the underlying biological problem. while, on the one hand, it is not that straightforward to come up with a proper objective criterion in biological problems, on the other hand, choosing the objective based on mathematical tractability with disregard of its meaning will not pass muster. in this paper, which is intended as a review and discussion, using mathematical models for cancer treatments as the main vehicle of presentation, we discuss the main aspects that enter into the modeling of biomedical problems as optimal control problems. we give some attention to the formulation of the dynamics (such as growth and interaction terms for the population) and treatment aspects, but our emphasis will be on the formulation of the objective to be minimized. we stress the need for a meaningful interpretation of the objective. this should be related to side effects of the treatment in models for cancer therapies and/or to the actual cost. yet, such costs are not always easily quantifiable. just think of 'social distancing' during an epidemic and its effects on the economy. using examples from our work, we illustrate how information from the underlying biology or medically relevant limitations can be used to formulate the optimization criterion so that optimal solutions then give meaningful information for the underlying biomedical problem. we point out some pitfalls to be avoided, but also make useful simplifications in the modeling, both in the dynamics and the objective. in this section, a general model is formulated which represents a class of dynamical systems common in the modeling of biomedical therapies. these models are affine in the controls u ∈ r m with states x ∈ r n + . the components x i , i = , . . . , n, of the state x are nonnegative numbers which, in a typical example, represent volumes or cell counts of various populations (compartments) of cells relevant to the underlying biomedical condition or, in epidemiological models, population counts in various stages of the disease. the controls u j , j = , . . . , m, stand for possible external interventions and strategies such as drug therapy in cancer treatments or vaccination efforts in epidemiological models. mathematically, the general structure we consider in this paper is of the form journal of optimization theory and applicationṡ with f and g typically nonlinear vector fields called the drift and control vector fields, respectively, and u the control set which represents restrictions on the size of the controls. the drift vector field f describes the underlying dynamics of the system without any outside interventions. it generally consists of various terms which should include the following aspects: growth terms of the populations as well as transition and interaction terms between the various compartments. these terms describe the mechanisms behind the increase in all or some of the populations in the compartments that define the state. these mechanisms can be modeled in various ways taking into account whether or not such growth saturates or not. the simplest model for a single population x ∈ r is given by exponential growth, x = ax, with a a constant representing the growth rate. the same expression with a < describes a basic death term of the population and, more generally, these two processes can be combined with a describing the net effect. while this is an adequate model for some phases of the dynamics, often used in short-term models or in epidemiology, over longer time periods saturation effects need to be taken into account. here, the most common models are logistic growth,ẋ = ax − x k , with k denoting a fixed carrying capacity of the population. another saturation type model, which has been verified empirically as the model for late-stages in breast cancer [ , ] , is gompertzian growth of the formẋ = −ax ln x k . logistic growth is often preferred because of its simpler mathematical structure. other growth models exist, but are less common (e.g., see [ ] ). transition terms model the exchanges between populations. for example, in cellcycle specific models for cancer chemotherapy [ ] , quiescent and proliferating cells are distinguished and proliferating cells move through growth phases, synthesis and mitosis, all represented by different coordinates of the state vector x. similarly, in models including drug resistance, random changes from sensitive to resistant cells occur which, in cases of cancer chemotherapy, also may be reversible. in epidemiological models, transitions between sensitive, exposed, infectious and recovered individuals are crucial to understand the spread of the disease. all such transitions are generally modeled by linear transition rates of the forṁ with α i ≥ representing the outflow from the ith compartment and β i j ≥ for i = j representing the inflow from the jth into the ith compartment. cells in various compartments stimulate and/or inhibit cells in other compartments. often these interactions are modeled by the law of mass actions leading to products of terms representing the cell counts or volumes in various compartments. this generates expressions of the form x i x j or, more generally, expressions which include powers of the variables. the simplest structure shows up in epidemiological models when susceptible and infectious populations s and i interact generating an outflow of the form β s i from the susceptible compartment which becomes an inflow to the infectious population i with β accounting for the probability of such interactions. powers arise, for example, in a model for angiogenic signaling by hahnfeldt et al. [ ] where the interactions are between the tumor surface through which angiogenic inhibitors need to be released and the carrying capacity of the vasculature. if both tumor volume and the carrying capacity are modeled by volumes p and q, respectively, this generates an interaction term of the form p q. in a classical model by stepanova of tumor immune system interactions [ ] , the fact that the immune system is able to control small tumors, but increasingly becomes overwhelmed by large tumors is modeled by an interaction term of the form α x − βx y where x represents the tumor volume and y a non-dimensional, order of magnitude variable related to the actions of the immune system called the immunocompetent cell density. here, x = β corresponds to a threshold beyond which the immunological system becomes depressed by the growing tumor. the coefficients α and β are used to calibrate these interactions and collectively describe state-dependent interactions between the cancer cells and the immune system. limiting effects of the interactions between various compartments can be taken into account by so-called michaelis-menten terms. as an example, in a model for cml (chronic myeloid leukemia) [ ] which includes proliferating cancer cells p and effector cells e of the immune system, this leads to terms of the type p max · p pc + p · e with p max denoting the maximum possible effect and pc the concentration of proliferating cancer cells for which half of the maximum effect is realized. similar terms describe stimulating or inhibiting effects of such interactions depending on the nature of the variables. important aspects of the uncontrolled dynamics are multi-stability and the geometry of the regions of attraction of locally stable equilibria. we illustrate these features using two classical models for tumor growth and its interactions with the tumor micro-environment: the model for angiogenic signaling by hahnfeldt et al. [ ] and stepanova's model for tumor immune-system interactions [ , , ] . these models also serve as illustrations for the general elements of the dynamics described above. (a) angiogenic signaling in this model by hahnfeldt et al. [ ] , the spatial aspects of the underlying consumption-diffusion process that stimulate and inhibit angiogenesis are incorporated into a non-spatial -compartment model with the primary tumor volume, p, and the carrying capacity of the vasculature, q, as its principal variables. the dynamics consists of two odes that describe the evolution of the tumor volume and its carrying capacity which are given bẏ thus, a gompertzian growth term is used to model tumor growth. the carrying capacity q and tumor volume p are balanced for p = q, while the tumor volume shrinks for inadequate vascular support (q < p) and increases if this support is plentiful (q > p). different from traditional approaches, the carrying capacity is not considered constant, but becomes a state variable whose evolution is governed by a balance of stimulatory and inhibitory effects. the term bp represents the stimulation of the vasculature by the tumor and is taken proportional to the tumor volume. the term dp q models the inhibition of the vasculature by the tumor which, as mentioned above, is represented as an interaction term between the tumor surface and the vasculature. the coefficients b and d are mnemonically labeled for birth and death, respectively. the last term −μq simply represents a natural death term related to the vasculature. this model has a unique equilibrium at which is globally asymptotically stable (relative to the natural state-space d = {( p, q) : p > , q > }) [ ] (see fig. ). the dynamics has a strong differentialalgebraic character with the q-dynamics of the vasculature fast and the p-dynamics for the tumor volume slow. essentially, the system follows the corresponding null-cline (defined by the equationq = ) into the unique equilibrium point. for realistic values of the parameters, this equilibrium is not biologically viable and therapy is needed to lower or even eradicate this equilibrium point. in the later case, the objective then becomes to drive the state of the system toward the singular point ( p, q) = ( , ). (b) tumor immune system interactions in this by now classical mathematical model of stepanova [ ] , two ordinary differential equations formulate the aggregated interactions between cancer cell growth and the activities of the immune system during the development of cancer. precisely because of its simplicity-a few parameters incorporate many medically important features-the underlying equations have been widely accepted as a basic model. the main features of tumor immune system interactions are aggregated into just two variables, the tumor volume, p, and the immunocompetent cell densities, r , a non-dimensional, order of magnitude quantity related to various [ ] , a fast growing cancer. the almost horizontal segments indicate a strong differential-algebraic character with trajectories eventually following theq-nullcline defined byq = types of immune cells (t-cells) activated during the immune reaction. the resulting dynamics takes the following form: with f a tumor growth function. greek letters denote constant coefficients which model the main interactions of the tumor with the immune system. various organs such as the spleen, thymus, lymph nodes and bone marrow, each contribute to the development of immune cells in the body and the parameter γ in eq. ( ) models a combined rate of influx of t-cells generated through these primary organs; δ is simply the rate of natural death of the t-cells. the first term in this equation models the proliferation of lymphocytes. for small tumors, it is stimulated by tumor antigen and this effect is taken to be proportional to the tumor volume p. large tumors suppress the activity of the immune system, mostly because of a general suppression of immune lymphocytes. this feature is expressed in the model through the inclusion of the term −β p . thus, /β corresponds to a threshold beyond which the immunological system becomes depressed by the growing tumor. the coefficients α and β are used to calibrate these interactions and collectively describe state-dependent influence of the cancer cells on the stimulation of the immune system. the first equation, ( ), models tumor growth with ξ a tumor growth coefficient that has been separated from the qualitative behavior of the growth function f. the second term, −θ pr , models the beneficial effects of the immune system reaction on the cancer volume and θ denotes the rate at which cancer cells are eliminated through the activity of t-cells. the dynamical behavior of this system is fundamentally different from the model for angiogenic signaling. for a typical set of parameter values, the dynamics is multi-stable table and varying growth functions f. the following growth functions were used in the respective panels: a gompertzian growth: c generalized logistic growth with exponent : , and d exponential growth: f e ( p) ≡ . the points ( p b , r b ) and ( p m , r m ) are the locally stable benign and malignant equilibria, respectively, and ( p s , r s ) denotes the unstable saddle point. the dashed red curve is the stable manifold of this saddle and the system has both locally asymptotically stable microscopic and macroscopic equilibrium points as well as an unstable saddle point. figure shows phase portraits of the system for the parameter values listed in table for four different growth models which qualitatively all exhibit the same multi-stability. at the microscopic equilibrium point, the tumor volumes are small and immuno-competent cell densities are upregulated. this corresponds to a situation when the immune system is controlling the tumor. we denote the corresponding equilibrium point by ( p b , r b ) and call it benign. the macroscopic equilibrium point is characterized by more than tenfold higher tumor volumes and depressed immunocompetent cell densities. in these solutions, the tumor has suppressed the immune system and it almost reached its carrying capacity. we denote the corresponding equilibrium point by ( p m , r m ) and call it malignant. both of these equilibria are locally asymptotically stable, ( p b , y b ) a stable focus and ( p m , r m ) a stable node. we call the regions of attraction associated with the benign and malignant equilibria the benign, respectively, the malignant region. for the model with an these phase portraits represent a snapshot of the true dynamics: a stationary situation for fixed parameter values. because of unmodeled aspects of the dynamics and/or random events, however, this situation will constantly change in time. thus, it is intuitively clear that a benign equilibrium solution can be disrupted by events affecting the immune system which were not included in the mathematical modeling which then may lead to a transition of the state into the malignant region. how likely this becomes is related to the size of the region of attraction of this equilibrium: if the benign region is small, even minor events may bring up the disease, whereas the immune system may be able to control the disease if this region is large. while there exist good reasons to believe that the immune system is able to control some tumors initially, over a longer period of time, the neoplasm will develop various strategies to evade the actions of the immune system (immuno-editing) and this allows the tumor to recommence growing into clinically apparent tumors and eventually reach its carrying capacity [ ] . tumor-immune system interactions thus exhibit a multitude of dynamic properties that include persistence of both benign and malignant scenarios. from a practical point of view, the question then becomes how to move an initial condition that lies in the malignant region into the benign region. this requires therapy and can naturally be formulated and analyzed as an optimal control problem. besides the underlying dynamics represented by the drift vector field f, the other essential ingredient in biomedical problems is a mathematical model for outside inter-ventions. these comprise therapies aimed at curing a patient in cancer treatment or actions to limit the spread of an epidemic. within the general structure ( ) of the model considered in this paper, the effects of these actions are modeled linearly through the control vector fields here the control variables u i , i = , . . . , m, represent various therapy options such as chemotherapy, radiotherapy, immunotherapy in mathematical models for cancer treatments and/or other available actions to influence the state of the system such as, for example, social distancing measures in epidemiological models. the components . . m, model the effects which the jth intervention modality u j has on the ith compartment x i of the state. generally, the number m of different modalities for outside interventions in a biomedical problem setting is small and m typically lies in the range of m = , , . even for the current covid- pandemic, in essence, all efforts to limit the spread of the pandemic are related to social distancing measures in an attempt to lower the probabilities of infection. if available, vaccination would be a different second intervention strategy, but, clearly, the overall number of options is not very large. similarly, in cancer therapy, the number of possible qualitatively different treatments options is small. if one ignores surgery, as this is an option that would be pursued at the onset prior to any time frame that typically would be modeled mathematically, the main modalities are chemotherapy, radiotherapy and immunotherapy. if chemotherapy is given through a cocktail of drugs (and this is the common procedure), then it is still typically advantageous to model the effects of the combination of all the drugs together and one may have m = , while the case m > often is reserved for true combination therapies that combine, for example, chemo-or radiotherapy with antiangiogenic treatment. thus, the typical scenario is that the number m of controls in the system is small. the particular mathematical structure considered in eq. ( )-linear in u-clearly does not cover the full range of possibilities, but it is worth emphasizing that this is by far the most common, most important, and, in many aspects, most natural structure. essentially, whether or not a control affine model ( ) is valid depends on the choice of what the controls represent. if the controls are tied in with the effects of treatment (e.g., the control represents the concentration of an agent rather than its dose rate), then this model may not be appropriate. if, however, the controls u i are related to the administration of the therapies (dose rates) and are separated from their effects, these models will be linear in the controls. below we shall discuss in more detail mathematical models for drug therapies, but we want to mention at least in passing the main model for radiotherapy, the so-called linear quadratic model. in this model, the probability of cell survival is represented in the form exp −α d − β d where d denotes the total radiation dose and α and β are radiosensitive parameters. briefly, the rationale for the model is as follows [ , ] : radiation causes abnormalities (lesions) that correspond to ruptures of the molecular bonds on the double-stranded dna. this damage is made up of two components: (i) simultaneous breaks in both dna strands that are caused by a single particle and (ii) adjacent (both in location and time) breaks on separate strands. while a double-strand break leads to a loss of proliferative abilities, a single-strand breakage is not considered lethal since dna has the ability to repair it. only if a second adjacent break occurs on the other strand before the first one can be repaired, this will lead to loss of proliferation properties. both scenarios (i) and (ii) are considered lethal in the sense that the cell is no longer able to proliferate. the first situation leads to a linear term in the cell survival model, while the second one contributes quadratic terms. this is accounted for by the parameters α and β which are called the lq parameters. repair of dna damage generally is incomplete. once this is taken into account as well, then the surviving fraction of cancer cells can be represented in the form with u denoting an arbitrary, time-varying dose rate, u = u(t) ≥ , over the therapy interval [ , t ] and ρ the tissue repair rate. the total dose d is given by d = t u(t)dt. this model can be written in a control-affine form [ , ] as follows: here, p once more represents the tumor volume and the equation for r models the temporal effects of tissue repair; α and β are the lq parameters. the specific parameter values depend on the tissue treated, and there will be separate equations modeling the effects on cancerous and healthy tissue. the control u represents a time-varying fractionated radio-therapy dose rate. the linear model in u represents the way therapy is administered, and this almost always is adequately modeled by a linear term. the quadratic effects of radiotherapy are subsumed at the expense of introducing the extra state variable r . similarly, while the actions of different treatment modalities clearly are interrelated, this concerns their effects, not the way the therapies are administered. if chemotherapy is considered with multiple drugs, then synergistic or antagonistic effects which cannot be modeled linearly become important. aside from the fact that such interactions often are far from being understood, it also matters what the control u represents. if the control represents drug concentrations, then such interactions enter into the model through the controls and a linear model is inadequate. on the other hand, if the controls represent the dose rates of the various agents, then the concentrations of these agents merely become additional states and these interactions enter into the drift vector field f of a generally already nonlinear dynamics. this then preserves the control linear structure ( ) . in essence, if the controls u i , i = , . . . , m, represent the administration of the therapies (dose rates) and as variables are separated from the effects of the actions (which, for example, depend on the concentrations), then a model which is linear in the controls is not only adequate, but is the correct one. we expand further on this for the case of chemotherapy and, as a matter of simplicity of the presentation, focus on the case of a single control u. pharmacodynamics is the branch of pharmacology which quantifies the effects which a drug has at concentration c. according to the law of mass action, the speed of a chemical reaction is proportional to the product of the active masses (concentrations) of the reactants. if a drug is administered, in an ideal situation it has been postulated that cell death under cancer drugs follows first-order kinetics [ ] , i.e., the decrease in the number of cancer cells per unit of time is proportional to the number of cancer cells with the rate depending on the concentration of the anti-cancer drug. any functional relation of the form e = s(c)x with x denoting the cells in a compartment on which the drug is acting (e.g., tumor cells, immune system, healthy cells, etc.) and the coefficient s(c) representing the concentration dependent killing rate of the drug is considered a pharmacodynamic model. a linear model of the form s(c) = ϕc with ϕ a positive constant, the so-called linear log-kill hypothesis based on skipper's paper [ ] , is the most commonly used form. incorporating this structure into a standard growth law results in the following growth model under chemotherapy, if the concentration c is considered the control u in the system, u = c, then the linear log-kill model fits the general linear form ( ) . the product represents an interaction term as discussed in sect. . . . such terms can be both inhibiting (minus sign) and stimulating (plus sign). clearly, in the example considered above, the action is inhibiting as the agent is cytotoxic and kills the cells. on the other hand, in stepanova's model the action of an immune stimulatory agent (such as interleukin- , il- , representing immunotherapy) would be included through the addition of a positive term +ρru into eq. ( ) . while a linear model s(c) = ϕc is applicable over a specified range of the concentration c, it is not valid for low or high drug concentrations. this simply stems from the fact that most drugs have little to no effect if their concentrations are too low and, on the other hand, their effectiveness saturates at high concentrations. if side effects allow, drugs are preferably given in the upper range of this dose-effect curve and an e max model of the form describes the effect of "fast" acting drugs more accurately for high concentrations. here, ec denotes the concentration at which half of the maximum effect e max is realized. sigmoidal functions also capture the behavior at lower concentrations. in these cases, if the drug concentrations are used as the control, after normalization this leads to terms of the form u +u which no longer fit the proposed structure ( ). this, however, is merely caused by using the concentrations as the controls. it can easily be removed by changing the model to treat the dose rates of the drug administration as the control. the second major aspect of drug delivery is pharmacokinetics. pharmacokinetic equations model the time evolution of a drug's concentration in the blood plasma. the standard model that describes the concentration c = c(t) of the drug in the bloodstream in response to a continuous dose rate u = u(t) is one of exponential growth and decay given byċ with ρ the clearance rate of the drug. more generally, if the concentrations in the bloodstream are separated from the concentrations in a peripheral compartment (such as tissue or an organ of interest) and the interactions between those compartments are taken into account, a two-dimensional linear system with positive eigenvalues is used. higher-dimensional compartmental models are used less frequently, but arise, for example, if the peripheral compartment is divided further into a 'shallow' and 'deep' compartment. for example, a -compartment model is often used for insulin pumps [ ] . all these models, however, are described by linear time-invariant systems with real eigenvalues. if one therefore introduces the drug dose rates as the controls, then the associated drug concentrations c become state variables and thus become an integral part of the modeling of the uncontrolled dynamics that enters the form of the drift vector field f. in particular, all the nonlinearities that arise because of saturation effects or because of synergistic properties of drug administration are subsumed in the drift vector field f, which, as the example for angiogenic signaling shows, may already be a highly nonlinear vector field to begin with. actual drug administration, however, is described by linear models. once medical professionals have decided on a course of action, a number of natural questions need to be answered: ( ) how much of the agent should be given? ( ) when and how often should drugs be given? ( ) in what order should the treatments be applied? all these questions can naturally be answered within the framework of an optimal control problem. clearly, we are only dealing with a mathematical model and no claims are made that the real problem could be solved in this way. but interesting and useful information can be obtained. to give a concrete example, chemotherapy needs the tumor vasculature to deliver cytotoxic agents that kill the tumor cells. it therefore was conventional wisdom to first give chemotherapy when antiangiogenic treatments and chemotherapy were combined in the treatment of cancer. this notion, however, was challenged by jain et al. [ , ] who argued that a normalization of the vasculature ("pruning") prior to the administration of chemotherapy is beneficial for the delivery of the cytotoxic agents and that it gives better results. these findings can be strongly supported through a mathematical analysis of the hahnfeldt model with both chemotherapy and antiangiogenic therapy [ ] . thus, while optimal control cannot provide the ultimate answers to the underlying problems, it can become a valuable tool in understanding these processes. the ultimate aim in biomedical problem formulations is to find a safe and reasonable strategy to solve the underlying medical problem. by adopting an optimal control formulation, we are aiming for what in a certain way can be considered the "best possible" solution. what this best possible solution should be, however, is often up to wide interpretations. it is here that the role of the objective functional chosen to minimize matters greatly-sometimes to the extent that this choice already preprograms the eventual solution. this choice then is the third essential aspect of the problem formulation. the following general form encompasses a wide range of possibilities: ( ) here, l is a sufficiently smooth function of the state called the lagrangian, the coefficients θ j , j = , . . . , m, are nonnegative weights and ϕ is a penalty function on the terminal state. the terminal time t can be fixed or free. the minimization is over a class u of admissible controls u which are locally bounded lebesgue measurable functions such that the associated controlled trajectory (x, u) satisfies all other constraints imposed on the system. these generally include control-constraints and terminal point constraints, but could also come from a wide variety of other restrictions imposed possibly by state-space constraints or other limitations inherent to the problem formulation. we discuss the significance of each of the terms starting with the controls. generally, and without loss of generality, we make the assumption that the controls represent nonnegative quantities (e.g., dose rates or concentrations). thus, the control term m j= θ j u k j (t) in the objective ( ) for k = or k = represents a weighted l -or l -norm, respectively. including such a positive and increasing term in the objective represents what in the engineering literature would be called a soft modeling of control constraints. clearly, medical treatments, especially the strong treatment options that need to be pursued in cancer treatment such as chemo-or radiotherapy, have significant side effects and these need to be taken into account in the modeling. including the control term in the objective to be minimized inherently limits the use of the controls. this represents an attempt to make a reasonable compromise between maximizing the tumor kill which requires to give a large amount of drugs and limiting the side effects of treatment (these are measured indirectly through the integral of the control) which requires to limit this amount of drugs. whether the optimal control does this adequately, then becomes an after-thought that always needs to be checked and, if the optimal solution is still inadequate, the weights need to be adjusted. if the weights on the drugs are too small, optimal solutions will simply give drugs all the time at full dose; if the weights are too high, no drugs will be given. it is clearly necessary to calibrate the weights to obtain meaningful results. such back-and-forth approaches are quite common in engineering and they simply reflect the fact that the form of the objective, and especially the weights that appear, are variables of choice in such problems. generally, and this is the typical application of optimal control in engineering, various choices of the weights will be tested until a solution has been found which appears adequate, possibly also with respect to other criteria which were not included in the mathematical model. the choice of taking an l -or l -norm, however, is not irrelevant. generally speaking, an l -formulation is to be preferred from a modeling perspective while the l -formulation offers significant mathematical and numerical advantages. in chemotherapy, there exists a clear interpretation for the integral t u(t)dt related to the total dose given regardless of whether u represents the dose rate of the drug or its concentration. this is an essential pharmacological quantity also referred to as the auc (area under the curve), and it plays a major role in interpreting the overall efficacy and side effects of a drug. thus, making the choice of an l -formulation as the penalty of the control leads to a realistic modeling. the handicap of an l -type approach lies with the fact that the mathematical analysis generally becomes difficult. optimal solutions need to be synthesized from concatenations of bang and singular controls (and we shall discuss this more in the next section) and reliable numerical methods to solve such problems do not exist. it is for this reason-and in our opinion, for this reason only-that the use of an l -criterion in the literature is wide-spread. for, in this case, the application of the necessary conditions for optimality of the pontryagin maximum principle [ ] [ ] [ ] [ ] leads to a closed form formula for the control as a function of state and multiplier that can easily be solved numerically. this, however, does not guarantee that a numerically computed solution is optimal as only extremals (pairs of controlled trajectories and multipliers which satisfy the first-order necessary conditions for optimality) are obtained, a fact often understated by many researchers. indeed, the projection from the space of extremals into the state space (i.e., projecting out the multiplier) can haveand often does have-singularities, the equivalent of the well-known conjugate points from the calculus of variations. for problems employing an l -type objective, higherorder tests for local optimality are given in [ , chapter ] or [ ] and are not difficult to apply, but generally this is not done in the literature. our main objection to the use of an l -criterion in the controls, however, does not lie with this mathematical aspect, but simply with the fact that for biomedical problems it seems rather impossible to give a satisfactory justification for such a functional form as the meaning of the phrase "systemic cost" employed in the vast literature is hard to capture. in chemotherapy, for example, not only does the quantity t u(t) dt not represent any pharmacologically meaningful quantity, but in addition, if one normalizes the controls to satisfy ≤ u i ≤ , as it is often done, then his strongly favors smaller partial doses over the higher full doses as half the maximum dose is only given a quarter of the weight of the maximum dose. thus, from the very setup, a bias is built into the optimal solutions to be computed which the underlying system does not have. cost is another questionable justification for an l -type objective as $ just is not a very meaningful quantity. it is certainly true that the cost need not be linear in u. this particularly holds in epidemiological models where the cost of vaccinating the first % of a population clearly is much smaller than the cost of vaccinating the last % of this population as much greater efforts will be needed to reach the tail end of the population. yet, while this clearly makes a point for some nonlinear relation, a quadratic term does not seem to be the answer to this. we still remark that it is often the typical scenario that optimal solutions for the l -type term (which necessarily are continuous) exhibit sharp rises from the lower control value u = to its maximum value u = u max mimicking optimal bang-bang controls which would be obtained by using an l -type term. indeed, from a numerical perspective, it makes sense to consider the problem when the control terms are taken in the form u +εu and then the limit as ε → is taken (if it exists). we note, however, that our interest in solving the optimal control problems is more in trying to obtain a better global understanding of the dynamics, i.e., the underlying problem, than to obtain one particular numerical solution (which is a much easier problem). as a rule, the parameter values in the model are uncertain and the solutions can be quite sensitive to these values (e.g., for stepanova's model) which limits the practical use of small numerical studies. we illustrate some of these effects with locally optimal solutions for a cell-cycle specific model for cancer chemotherapy due to swierniak [ , ] . in such models, the various phases of the cell-cycle-a quiescent phase g , a first growth phase g , synthesis s, a second growth phase g , and mitosis m-are clustered into compartments and the dynamics describes the transitions, i.e., the flow between these compartments. mathematically, the dynamics becomes a bilinear system of the forṁ with the state n i representing the average number of cells in the respective compartments and the matrices a and b j modeling the flow rates between the compartments. the controls represent cytotoxic agents which indiscriminately kill cells, cytostatic agents which block the transitions of cells through the cell-cycle and recruiting agents which entice cells to leave the quiescent phase and become active again so that it is possible to kill them. a typical form of the objective is given by with the coefficients nonnegative weights. figure compares locally optimal solutions for a -compartment model with the compartments n ∼ g /g , n ∼ s and n ∼ g /m for l -and l -type functional terms on the controls which represent a cytotoxic agent u and a cytostatic agent v [ , , ] . optimal controls for the l -type objective are bang-bang, i.e., switch between full dose therapies and rest-periods while optimal controls for the l -type objective are necessarily continuous. for an l -type objective, the optimal administration of the killing agent u is full dose initially followed with a rest period (and this is what medically makes most sense here), whereas for the l -type objective the continuity of the control generates decreasing lower dose administrations of the cytotoxic agent toward the end of the therapy interval. the total doses given in both cases are similar. for the blocking agent v, the optimal l -control clearly mimics the optimal l -control with steep continuous intermediate segments. the behavior of the controlled trajectories for both objectives is very much the same. the terminal values for the states corresponding to the l -and l -type objectives are close, and the reduction in the total cancer burden is from the initial normalization of c( ) = to c( ) = . for minimizing j and to c( ) = . for minimizing j . in both cases, the initial condition was chosen as the normalized steadystate of the uncontrolled dynamics (i.e., assuming no earlier treatment). computing the total dose of the drugs given for the l -optimal control, one sees, however, that while minimizing the quadratic objective leads to about a % improvement in the reduction of the tumor, this is at the expense of increasing the amounts of both the cytotoxic and cytostatic agents by about %. thus, higher side-effects are encountered. minimizing j would appear to be the preferred strategy. an alternative approach-and the one which seems to be preferred by medical practitioners-is to impose the total amount of drug (or radio therapy dose etc.) given a priori. the argument is that, to begin with, any mathematical model represents only a small view of the underlying problem and that, by sheer necessity of the complexity which, for example, cancer therapy is, many aspects need to be neglected. thus, the point of view is taken that, based on other assessments such as the side effects of the therapy, the total amount of drug to be given has been determined a priori, here, then the question simply becomes the following one: given this amount of drug, how can it be administered in the best possible way? mathematically, this amounts to adding the trivial equationẏ = u, y( ) = , to the dynamics and the terminal condition y(t ) ≤ a is imposed as an isoperimetric constraint. this approach, for example, was taken in solving hahnfeldt's model for antiangiogenic therapy as an optimal control problem [ , ] . in such a case, no control related term will be included in the objective. from a modeling perspective, the use of a hard constraint on the controls seems to be more adequate. mathematically, however, there are close relations between the necessary conditions for optimality for both formulations (either as a soft or hard constraint) and it makes little difference if one or the other formulation is chosen. the lagrangian l and the penalty term ϕ allow to judge the impact of the therapy on the state. including the lagrangian l in the objective can also be viewed as incorporating a soft constraint on the state. for example, in cancer therapies, it is clearly important to limit the size of the tumor, or, in another indirect measurement of the side effects, prevent that the level of the bone marrow becomes too small. clearly, such restrictions could be modeled as state-space constraints imposing upper or lower bounds on some of the variables. for example, in [ ] an explicit upper bound in the form fig. examples of locally optimal controls (left) and their corresponding trajectories (right) for a cellcycle specific -compartment model for cancer therapy over a fixed therapy horizon (t = days) with lagrangian term l in the objective (top row, q = . ) and without lagrangian term in the objective (bottom row, q = ). the initial condition is the normalized value of the steady-state solution for the uncontrolled system is imposed on the total number of cancer cells n (t) in a cell-cycle specific model for cancer chemotherapy. here, however, a modeling approach of using soft constraints is preferred as the necessary conditions for optimality become more involved if statespace constraints are present. if explicit state-space constraints are included in the modeling, then, a priori, the associated multiplier is only known to be a positive radon-measure. although it can be shown that this measure is absolutely continuous with respect to lebesgue measure if the constraint is an embedded hypersurface (e.g., see [ ] ), the effort of dealing with a state-space constraint is much more involved than having a lagrangian present in the objective. a common pitfall in the modeling is not to include such a term. then, naturally, the emphasis of the minimization is put on the terminal time and quite often control actions will be delayed initially and be concentrated near the terminal time. once again, however, this is then not a feature of the underlying problem, but it has been imposed, possibly unwillingly, by the particular form of the objective that was chosen. figure illustrates this feature by comparing the optimal solutions for a -compartment cell-cycle specific model for cancer chemotherapy [ , , ] . the two compartments represent the clustered phase n ∼ g /g + s and n ∼ g /m of the cell cycle and the drug is a g /m-specific cytotoxic agent. the objective was chosen in the form for q = . and q = over a fixed week therapy period. optimal controls are bangbang with one switching. if only a penalty term is used, then the drug administration is all put to the end of therapy with an unacceptable spike in the tumor cells in between. the choice of the objective is often made in an heuristic ad hoc manner. sometimes, however, the underlying biology can be used as a guide. stepanova's model provides a beautiful illustration how this can be achieved. assuming that the parameter values are such that the dynamics is bi-stable with a benign and a malignant region, the practical aim of therapy then can be formulated as moving the state of the system from the malignant region into the region of attraction of the stable, benign equilibrium point while keeping side effects tolerable. assuming the controls are a strongly targeted cytotoxic agent u and an immuno-stimulatory agent v, this can be achieved using an objective functional of the following form: all coefficients are positive and the choice of the weights aims at striking a balance between the benefit at the terminal time and the side effects measured by the total amount of drugs given while it also guarantees the existence of an optimal solution by penalizing the free terminal time t [ , , ] . we discuss the significance of each penalty term in ( ) in detail: (i) the term ap(t ) − br(t ) at the terminal time is designed to induce a transfer of the system from the malignant into the benign region of the state space. as is seen in fig. , points with small tumor volumes and depressed immunocompetent cell densities lie in the malignant region and it may not suffice to simply minimize the tumor volume. rather, it is the geometric shape of the stability boundary between the benign and malignant region that matters. even for a low-dimensional dynamical system like stepanova's model, it is generally not possible to give a functional description of the separatrix which is the stable manifold of the unstable equilibrium point of the system. its tangent space, however, is easily computed, and it thus becomes a good heuristic to choose the coefficients a and b in the penalty term such that the minimization of this term corresponds to moving in the normal direction to this stable manifold from the malignant toward the benign region. alternatively, one could also choose these coefficients to move into the direction of the unstable eigenvector at the saddle point. for, this is the tangent vector to the path which uncontrolled trajectories will closely when in the benign region. in the formulation ( ) we have followed the first approach and v = (b, a) t is the stable eigenvector of the saddle point oriented so that both a and b are positive numbers (see fig. ). it follows from the geometry of the stable manifold that a and b will have the same sign and including in the objective a term of the form ap(t ) − br(t ) gives the correct direction to minimize in the objective. the level sets of this quantity are lines parallel to the tangent space of the stable manifold ) with some sample trajectories shown in blue. the dynamics has three equilibria: a locally asymptotically stable focus which is benign (shown as a small green circle), a saddle point (shown as a small grey circle) and a locally asymptotically stable node which is malignant (shown as a small red circle). the stable and unstable manifolds of the saddle point are shown as solid black curves. the tangent vector to the stable manifold of the saddle at the equilibrium point is labeled v = (b, a) t oriented so that both entries are positive of the saddle, and minimizing this quantity thus creates an incentive for the system to move into the benign region. (ii) the integrals of the controls, t u(t)dt and t v(t)dt, measure the total amounts of drugs given and, once again, represent a soft modeling of the side effects of the drugs. clinical data as to the severity of the drugs should be reflected in the choices for c and d. naturally, the specific type of tumor and stage of cancer will need to enter into the calibration of these coefficients. in a more advanced stage, higher side effects need to be tolerated and thus smaller values of c would be taken. (iii) the penalty term st on the terminal time makes the mathematical problem well-posed. this term, which can be written either under the integral or as a separate penalty term st , is somewhat unusual. it has to do with the nature of the underlying problem and the emphasis on the regions of attraction. the existence of the asymptotically stable, benign equilibrium point generates controlled trajectories that improve the value ap(t ) − br(t ) of the objective along the trivial controls u = and v = . if no penalty is imposed on the free terminal time, then this creates a "free pass" structure in which the value of the objective can be improved without incurring a cost. as a result, in such a situation an optimal solution may not exist. intuitively, the controls can switch to (u, v) = ( , ) immediately as the separatrix is crossed and then take an increasingly longer time as they pass near the saddle point with the infimum arising in the limit t → ∞. from a practical point of view, this behavior of course is not acceptable. mathematically, this behavior is easily eliminated by including a penalty term on the final time in the objective. this limits the size of the therapy interval and creates a mathematically well-posed problem. we discuss general properties of optimal controls for control-affine systems when the l -norm (k = ) is used on the controls and give examples of solutions for some of the problems mentioned earlier. we consider the optimal control problem to minimize the objective ( ) over all admissible controls u ∈ u subject to the dynamics ( ) and terminal constraints. necessary conditions for optimality of a controlled trajectory (x * , u * ) are given by the pontryagin maximum principle [ ] (for some recent references, see [ ] [ ] [ ] ). these conditions are formulated in terms of the (control) hamiltonian h defined as where λ ≥ and λ ∈ r n are multipliers which we write as a row-vector. the most important of the conditions of the maximum principle states that the optimal control u * for almost every time minimizes the control hamiltonian h pointwise over the control set u along the optimal controlled trajectory x * and the multipliers λ and λ, i.e., we have that for the problems considered here, the hamiltonian h is affine in the controls and for each component u i of u the control set is a compact interval. hence, the minimum condition splits into m separate scalar minimization problems that are easily solved. defining the functions Φ j : [ , t ] → r, it follows that the optimal controls satisfy the function Φ j is called the switching function corresponding to the control u j . a priori, the control u j is not determined by the minimum condition at times when Φ j (τ ) = . in such a case, all control values trivially satisfy the minimum condition and thus, in principle, all are candidates for optimality. however, the switching functions are absolutely continuous and if the derivativeΦ j (τ ) does not vanish, then the control switches at time τ between u j = to u j = u max j ifΦ j (τ ) = . such a time τ is called a bang-bang switch. on the other hand, if Φ j (t) were to vanish identically on an open interval i , then, although the minimization property by itself gives no information about the control, in this case also all the derivatives of Φ j (t) must vanish and this condition puts strong limitations on the controls. extremal controls for which the switching function vanishes identically over an open interval i are called singular, while the constant controls u j = and u j = u max j are called bang controls; controls that only switch between and the maximum control values are called bang-bang controls. we mention that higher-order necessary conditions for optimality of singular controls are available, the so-called generalized legendre-clebsch conditions (e.g., see [ ] ), that allow us to distinguish between minimizing and maximizing singular controls. if the control represents dose rates for the application of some therapeutic agent, then bang-bang controls correspond to treatment strategies that switch between maximum dose therapy sessions and rest-periods, the typical mtd (maximum tolerated dose) type applications of chemotherapy. singular controls, on the other hand, correspond to time-varying administrations of the agent at intermediate and often significantly lower dose rates. there is growing interest in such structures in the medical community because of mounting evidence that "more is not necessarily better" [ , ] and that a biologically optimal dose (bod) with the best overall response should be sought. hence, the question whether optimal controls are bang-bang or singular has a rather direct interpretation and its answer is of practical relevance for the structure of optimal treatment protocols. summarizing, solving an optimal control is tantamount to determining the correct sequences and switching times for the controls as concatenations of bang and singular segments. we close this review with two examples of optimal solutions and interpretations of these results. we consider the model ( )-( ) for angiogenic signaling with an antiangiogenic agent u and a chemotherapeutic agent v. modeling the effects of these controls using skipper's log-linear model and treating the concentrations of the agents as the controls gives the following controlled dynamics: we are taking the point of view that the available amounts of agents have been determined a priori. we add the trivial dynamicsẏ = u andż = v which keep track of the amounts of agents used and impose the isoperimetric constraints t u(t)dt ≤ y max and t v(t)dt ≤ z max at the end-point. the terminal time t is free. once side effects have been modeled in this way, our objective simply becomes to minimize the tumor volume, i.e., j (u) = p(t ). from a practical point of view, the optimal solution then simply answers the following question: given a priori determined amounts of agents, how can they best be administered in time to maximize the tumor reduction? for the monotherapy problem when z max = , i.e., no chemotherapy is given, we have given a complete global solution to this problem in form of a regular synthesis of optimal controls [ , ] . this solution specifies the optimal control as a feedback function of the state ( p, q, y) for arbitrary initial conditions. figure gives a twodimensional representation of this solution into the ( p, q)-space with p shown as the vertical and q as the horizontal variable. for a medically realistic initial condition (with the carrying capacity larger than the tumor volume), optimal controls start with a full-dose segment (shown as solid green curves in the figure) until an optimal singular arc s is reached. this segment is short in time. using the conditions of the maximum principle, the curve s can be described explicitly as [ , ] μ + dp then, and for a rather long time, the optimal controlled trajectory follows this singular arc s (shown as the solid blue curve) until all antiangiogenic agents are used up. because of after effects there still exists a small interval on which the tumor volume decreases further shown as a dashed green curve. we note that, and different from the impression given in the phase portrait, the times along the individual segments are as described above. the system has a strong differential-algebraic character and as a result the dynamics along the full and no dose segments occurring in the optimal synthesis is very fast generating the almost horizontal lines seen in fig. . optimal controls are strongly characterized by the singular control which follows a specific time-varying concentration of the agents which maintains an optimal relation between the tumor volume p and its carrying capacity q. it is the functional relation defined by the singular arc which provides ideal conditions for tumor shrinkage. the optimal solution for the monotherapy problem provides the mathematical foundation on which optimal controls for combination therapies with antiangiogenic treatments can be computed. this holds both for combinations with radiotherapy [ ] and chemotherapy [ , ] . here, we still discuss the latter case. below we state the optimal concatenation structure for a typical scenario which includes an initial condition when the carrying capacity significantly exceeds the tumor volume, when there is ample supply of antiangiogenic inhibitors (whose side effects generally are low) and a more limited total dose of chemotherapy (which generally has much higher side effects) to be given. in such a situation, optimal controls (u * , v * ) consist of concatenations of four segments of the following form: initially, no chemotherapy is given, while antiangiogenic drugs are given at full dose until the optimal singular arc s for the monotherapy problem is reached. at this point, administration switches to the optimal singular control for the monotherapy problem until a specific time τ is reached. at this time chemotherapy commences in a single full dose (mtd) session. in the medical literature this has been called the 'optimal therapeutic window' [ ] . also, because of the onset of chemotherapy, the formula for the optimal singular control adjusts and this is seen as a small kink in fig. which gives a sample of the time-evolution for optimal controls u * and v * . then, both controls are given until all available drugs are exhausted. in eq. ( ) we have formulated the controls for the case that the antiangiogenic inhibitors run out first, but this depends on the total amounts available and could also be reversed. figure illustrates the shape of the optimal controlled trajectory in ( p, q)-space. mathematical optimization of the combined therapy strongly supports the following two qualitative conclusions: (i) there is an optimal relation between the tumor volume p and its carrying capacity q along which the tumor decrease is maximized. this relation is given by the optimal singular arc s of the monotherapy problem. (ii) along this path there is an optimal point so that chemotherapy is given in one full dose mtd administration as this point is reached. these properties reflect the ideas of 'pruning' the vasculature and of a 'therapeutic window' [ , ] proposed in the medical literature. fig. illustration of the qualitative shape of optimal controls u * (left) and v * (right) for combination treatments with chemotherapy and antiangiogenic inhibitors for medically realistic initial values: after a brief initial period of full dose therapy, administration of the antiangiogenic agent follows the singular control until, at a specific time τ , chemotherapy commences. at that time, the dose of the antiangiogenic agent adjusts to the presence of the chemotherapy. as the dose of the antiangiogenic agent intensifies along the singular arc for the monotherapy problem, in the presence of chemotherapy which also has antiangiogenic effects, the dose increases in a small jump at time τ . the control follows the adjusted singular control until all inhibitors are exhausted. chemotherapy is given in one full dose segment commencing at time τ fig. "optimal therapeutic window": the red curve depicts the optimal controlled trajectory for the antiangiogenic monotherapy problem (the control is given by full dose therapy along the vertical portion and then follows the singular arc where the tumor volume decreases). chemotherapy then commences at an optimal time τ and the optimal trajectory is shown as solid blue curve. (the remaining optimal monotherapy trajectory is shown as dotted red curve.) when all antiangiogenic agents are exhausted, chemotherapy continues until all drugs have been administered. the junction point corresponds to the kink in the blue trajectory. chemotherapy still lowers the tumor volume, but as there are no more antiangiogenic agents given, the vasculature increases again we remark that the optimal controls in this model (and this is also the case in many other models where the concentration is taken as the control) are discontinuous. obviously, this a quirk of the modeling as concentrations are continuous functions. this discrepancy, however, is easily removed by adding a model for the pharmacokinetics of the drug which smoothes out the transitions between the segments while following the same structure. the simplified modeling with the controls representing concentrations has, from a mathematical point of view, the advantage of a lower dimensional state-space and, more importantly, the analysis of singular controls is simpler (c.f., [ , ] ). this makes the overall structure of solutions more tractable. our paper [ ] gives an in depth discussion of these aspects related to the pharmacometrics of the drugs. we conclude with an example of a numerically computed optimal solution for stepanova's model with strongly targeted chemotherapy u [ , , , ] . again we treat the concentration of the agent as the control and use skipper's log-linear model to describe the effect of treatment. this simply adds a term −κ pu to the dynamics ( ) of the tumor volume. choosing the objective functional in the form ( ) with n = (as we do not consider an immune boost), optimal chemotherapy protocols follow the concatenation structure s with representing a full dose segment, s denoting administration following a singular control and standing for a rest-period of the treatment. figure gives a characteristic example of a solution: the initial state z = ( p , r ) is characterized by a high tumor volume and depressed immuno-competent density close to the values of the malignant equilibrium point. in the figure, the unstable equilibrium point is marked with a green dot and the black curves crossing in it are its stable and unstable manifolds, respectively. initially, chemotherapy is given at full dose until the state of system has crossed over into the benign region. then it follows a singular arc where the concentration of the drug is dropped significantly through lower dose administrations. the full singular curve is indicated by a dotted red curve in the figure. finally, as the state of the system is in the benign region, chemotherapy is stopped and the uncontrolled dynamics (i.e., the actions of the immune system) lead to further reductions in the tumor volume. interestingly, toward the end, optimal chemotherapy protocols still apply a short chemotherapy segment. this indeed seems to be mirrored by what has proven a particularly effective administration protocol also in medical practice. if the effect of an immune boost is incorporated into the system, this structure is generally preserved. figure gives an example of an optimal control for the combination therapy with the chemotherapeutic agent shown in red and the immune boost in green. essentially, the immune boost is only given toward the end to steer the system into a better terminal state. this figure also clearly shows the lowering of the dose rate for the cytotoxic drug along the singular segment. it has not been our aim to discuss numerical methods in optimal control in this paper. for completeness sake, however, we include some brief comments on the methods that were used by us and our co-workers to arrive at the solutions shown in this paper. the initial point, all junctions (where the control switches), and the terminal point of the optimal trajectory are marked by black dots. starting at z = ( p , r ), initially full dose chemotherapy is used. along that trajectory, the system enters from the malignant region (below the stable manifold of the saddle point) into the benign region (above the stable manifold of the saddle point). the stable and unstable manifolds of the saddle point (marked by a green dot) are the black curves which intersect transversally in the saddle point. the singular arc is the u-shaped curve shown as solid green segments where the singular control is admissible and as dotted red segments where the singular control is inadmissible. once the optimal controlled trajectory meets the singular arc, the control switches to become singular and the trajectory follows the singular arc (at much reduced dose rates, c.f., fig. ) . then, at a certain time, chemotherapy stops and the trajectory follows the dynamics of the uncontrolled system approaching, and ultimately tracing, the unstable manifold of the saddle point. as the state lies in the benign region, this leads to an overall improvement of the objective value. the optimal control then ends with another brief full dose chemotherapy session to reach the optimal terminal point the problem is simpler if an l -type objective is employed in the control as this makes the hamiltonian h of the optimal control problem strictly convex and optimal controls are continuous. in this case, standard shooting methods can be applied to the combined system of dynamics and adjoint equations to compute numerical extremals. their local optimality is easily verified or excluded by computing conjugate points using a second-order approach which tests the existence of a solution of the associated matrix riccati differential equation as described in [ ] . other approaches are based on discretizations or pseudo-spectral methods (e.g., see [ ] [ ] [ ] ). for the solutions shown in sect. . we used gpops (general pseudo-spectral optimal control software), an open-source matlab optimal control software that implements the gauss hp-adaptive pseudospectral methods (http://www.gpops.org/, [ ] ). these methods approximate the state using a basis of lagrange polynomials and collocate the dynamics at the legendre-gauss nodes [ ] . the continuous-time optimal control problem is then transformed into a finite-dimensional nonlinear programming problem that is being solved using well-known and standard algorithms. fig. . the maximum chemotherapy dose rate (vertical axis) has been normalized to . as can be seen, the time-intervals when chemotherapy is at full dose are brief and the optimal dose rates drop significantly along the much longer singular segment. the rest-period is the most dominant time-period (almost units out of t = ) problems with an l -type objective which contain optimal singular arcs are more involved numerically as the singular control is only optimal on an embedded submanifold of positive codimension. such a structure is inherently difficult to locate numerically. various algorithms, including commercial programs, we tried were simply unable to deal with this situation. for the antiangiogenic monotherapy problem (see fig. ) the complete optimal synthesis was determined by us theoretically by analytical means [ ] . the computation of optimal controlled trajectories then amounts to the 'evaluation of a function' and is a matter of seconds (c.f., also [ ] ). for the combination therapies, based on the monotherapy results, the computations were made by maurer. states, controls and adjoint variables were computed using discretization methods which transcribed the optimal control problem into a large-scale nonlinear programming problem which was implemented in the framework of the applied mathematical modeling programming language ampl and is linked to various optimization codes. either the interior-point method ipopt based on [ ] or the optimization code nudocccs developed in [ ] in conjunction with the arcparametrization method [ ] were used. on the other hand, if optimal controls are bang-bang (such as for the cell-cycle specific methods for chemotherapy), the numerical problem is simple and basic algorithms which minimize over the switching times (e.g., gradient procedures [ ] ) can be used. a verification of second-order conditions for optimality can easily be built into this algorithm [ ] . in his paper, we formulated an optimal control problem for a general dynamical system which is nonlinear in the state and affine in the controls. the presented framework encompasses a number of models for treatment of biomedical problems, especially cancer therapies, as well as the more common models in epidemiology. the controls account for either therapeutic or epidemiological interventions. the terms in the cost functional represent various aspects and constraints that need to be taken into consideration when the dynamics is considered as an optimal control problem. we discussed, and using examples from our own work illustrated how the solutions to these problems share certain common characteristics, what differences can be expected to appear and, most importantly, how the choice of the objective affects the form of the solutions both quantitatively and qualitatively. specifically, it has been emphasized that omitting some important terms in the objective, like, for example, penalties on the tumor size during therapy-not an uncommon procedure in the literature-may lead to dubious solutions which indicate to wait and administer cancer treatment only toward the end of the therapy period. clearly, often mathematical features of the solutions (e.g., continuous versus discontinuous protocols for the dosage of a drug) are preprogrammed through the choice of the objective functional. this is acceptable, provided one is aware that this is an outcome of the mathematical formulation and not an indication of properties of the underlying dynamical system or biology. the purpose of this article was to review and somewhat organize and illustrate work done by us on this topic as well as to point out some common pitfalls 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chemotherapy with a stongly targeted agent for a model of tumor-immune system interactions with generalized logistic growth direct trajectory optimization and costate estimation via an orthogonal collocation method the chebyshev-legendre collocation method for a class of optimal control problems zero-propellant maneuver guidance user's manual for gpops: a matlab package for dynamic optimization using the gauss pseudospectral method optimal and suboptimal protocols for a class of mathematical models of tumor anti-angiogenesis on the implementation of an interior-point filter line-search algorithm for large-scale nonlinear programming sqp-methods for solving optimal control problems with control and state constraints: adjoint variables, sensitivity analysis and real-time control optimization methods for the verification of secondorder sufficient conditions for bang-bang controls a gradient method for application of chemotherapy models publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are grateful to four anonymous referees of this paper for their useful comments which helped us improve our presentation. all data generated or analyzed during this study are included in this published article. key: cord- - irru authors: pazos, f. a.; felicioni, f. title: a control approach to the covid- disease using a seihrd dynamical model date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: irru the recent worldwide epidemic of covid- disease, for which there is no vaccine or medications to prevent or cure it, led to the adoption of public health measures by governments and populations in most of the affected countries to avoid the contagion and its spread. these measures are known as nonpharmaceutical interventions (npis) and their implementation clearly produces social unrest as well as greatly affects the economy. frequently, npis are implemented with an intensity quantified in an ad hoc manner. control theory offers a worthwhile tool for determining the optimal intensity of the npis in order to avoid the collapse of the healthcare system while keeping them as low as possible, yielding in a policymakers concrete guidance. we propose here the use of a simple proportional controller that is robust to large parametric uncertainties in the model used. the novel sars-cov- coronavirus, which produces the disease known as covid- , was first reported on december in wuhan, province of hubei, china. with amazing speed it spread to the majority of the countries in the world. the outbreak has been declared as a public health emergency of international concern by the world health organization (who) on jan. , and as a pandemic on march . at the moment, there is no vaccine against this virus or effective medicines to cure the disease. health systems only try to mitigate its consequences to avoid complications and fatal outcomes. this disease showed a great capacity of contagion and high fatality rates (see updated reports in [wm ] ). patients affected by this disease experience a number of symptoms, not all clearly identified at the moment, but which are mainly cough, breathing difficulties, fever, loss of taste and smell and extreme tiredness. frequently, patients develop a form of viral pneumonia that requires hospitalization and artificial mechanical ventilation in intensive care units. the large number of patients affected by this disease threatens to collapse public health systems, increasing the fatality rates by lack of available health assistance. in this context, is very important to predict the trend of the epidemic in order to plan effective strategies to avoid its spread and to determine its impact. as the contagion is produced very easily by simple contact between people, several measures were adopted by the governments, public health systems and populations in order to reduce the transmission by reducing contact rates. examples of these measures, the so called nonpharmaceutical interventions (npis) adopted during this period include the closing of schools, churches, bars, factories, quarantine or physical-distancing policies, confinement of people in their homes, lockdown, among other social impositions that produce discomfort and clearly harm the economy. this goal sparked many articles and studies recently published on the epidemic behavior. a number of them are addressed towards determining a mathematical model that represents the dynamics of different agents involved in a population affected by the disease. the dynamic described by the model aims to make possible to answer crucial issues, such as the maximum number of individuals that will be affected by the disease and when that maximum will occur, and makes key predictions concerning the outbreak and eventual recovery from the epidemic. this information allows to devise public policies and strategies to mitigate the social impact and reduce the fatality rate. the seminal work [flng + ] exemplifies and analyses different strategies to control the transmission of the virus. most of the models adopted to represent the dynamical behavior of the covid- are based on the sir model (see [abd ] and references therein). the sir model is a basic representation widely used which describes key epidemiological phenomena. it assumes that the epidemic affects a constant population of n individuals. the model neglects demography, i.e. births and deaths by other causes unrelated to the disease . the population is broken into three non-overlapping groups corresponding to stages of the disease: • susceptible (s). the population susceptible to acquire the disease. • infected (i). the population that has acquired the virus and can infect others. • recovered (r). the population that has recovered from infection and presumed to be no longer susceptible to the disease . a brief description of these compartments is given below. susceptible people are those who have no immunity and they are not infected. an individual in group s can move to group i by infection produced through contact with an infected individual. group i are people who can spread the disease to susceptible people. finally, an infected individual recovered from the disease is moved from the group i to the group r. some references (see, for example [lzg + , ssb ]) considers the group r as removed population, or closed cases, which includes those who are no longer infectious from recovery and the ones who died from the disease. the summation of these three compartments in the sir model remains constant and equals the initial number of population n . in order to describe better the spread of epidemics, many works (see, for example [kan , gv , shd , nes ]) adopted the seir model. in the seir model a fourth group denoted as exposed (e) is added between the group s and the group i: • exposed (e). the population that has been infected with the virus, but not yet in an infective stage capable of transmitting the virus to others. this compartment is dedicated to those people who are infectious but they do not infect others for a period of time namely incubation or latent period. other works (see [abd ] for example) consider an additional compartment at the end of the sir or of the seir model to distinguish between recovered and death cases: • dead (d). the population dead due to the disease. in argentina, the daily death rate is . − . at the moment, in the covid- disease is an open question if a recovered person can get re-infected. even though some cases were recently reported, the reinfection rate value appears to be statistically negligible based on early evidence. thus, these models become the sird or the seird models, respectively. other works as [tbt + ,lzg + ,ssb ] consider the existence of other groups seeking to match the models proposed with the numbers obtained from the actual covid- disease. the work presented in [gbb + ] deserves to be mentioned. this work studies the evolution of the covid- in italy, and proposes a model denoted as sidarthe, where the letters correspond to eight groups denoted as susceptible, infected, diagnosed, ailing, recognized, threatened, healed and extinct respectively. all of them are subgroups of those presented in the seir model. this model discriminates between detected and undetected cases of infection, either asymptomatic or symptomatic, and also between different severity of illness, having a group for moderate or mild cases and another one for critical cases that require hospitalization in intensity care units. the authors affirm that the distinction between diagnosed and nondiagnosed is important because nondiagnosed individuals are more likely to spread the infection than diagnosed ones, since the later are typically isolated, and can explain misperceptions of the case fatality rate and the seriousness of the epidemic phenomena. the fact of considering more groups in the sidarthe model than in the seir model allows better discrimination between the different agents involved in the epidemic evolution, as well as a better differentiation of the role played by each one. however, the fact of considering more groups implies the knowledge of more rates, probabilities and constants that determine the dynamics between the groups. many of these values are difficult to know in practice, as well as to estimate the population of some groups, such as ailing (symptomatic infected undetected). the authors choose these constants and quantities to match the model to the actual data. of course, in order to achieve the goal of better determining public policies, we believe that the existence of some of these groups in the model used is not necessary. in order to better guide the determination of public policies to mitigate the spread of the virus we propose the use of the control theory. control theory has been successfully implemented in several areas other than physical systems control, for which it was initially designed. for example in economics, ecological and biological systems, many works demonstrate the success of its implementation. of course, regardless of the area focused, a good control strategy depends on the adequate modeling of the dynamical system to be controlled. the proposal to use control in this epidemic is not new. it has been first presented in [shd ] . in this work, the authors use the seir model to show that a simple feedback law can manage the response to the pandemic for all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . maximum survival while containing the damage to the economy. however, the authors illustrate with several examples the benefits of using feedback control, but they do not present the mathematical control laws as well as they do not prove the convergence of the trajectories in the closed loop system. examples are implemented by mean of several computational experiments which illustrate the different strategies proposed. we propose here the use of a simple proportional controller, a standard tool in control theory, to calculate the control action. this variable guides how to determine npis in order to avoid the collapse of the health system while reducing the damage to the society and the economy that npis inevitably produce. this section is addressed to model adequately the disease. a suitable model should avoid making unnecessary classifications in order to obtain key data on the behavior of the epidemic. these data include number of deaths, maximum number of infected people, time at which the maximum infection rate will occur, among other information useful to prevent and reduce the damage produced by the outbreak. the seir model assumes that exposed people have been infected but are not able to transmit the virus before a latency period. we will consider that those people continue to be in the susceptible group s, whereas we consider the group e as people who have been infected but have no symptoms yet and are capable of transmitting the virus. of course, part of this group will present symptoms after an incubation time (moving to the group i) and another part will remain asymptomatic. asymptomatic people who have been diagnosed as positive also will be considered in the group i, so this group includes all known positive cases, symptomatic or not. in addition, a critical issue is the number of infected people who need hospitalization, because the public policies must try to keep this number lower than the capacity of the health care system in order to avoid its collapse. thus we define an extra group: • hospitalized (h). the infected population who need hospitalization. in the group h we do not differentiate between people hospitalized in mild condition and those in intensive care units (icus), despite the fact that the number of people in the last subgroup is a critical problem due to an even more limited capacity in icus. figure : rate processes that describes the progress between the groups in the seihrd model. we also consider the population number n as a constant, as the seir model does. the progression of this epidemic can be modeled by the rate processes described in fig. . the proposed seihrd model for the spread of the covid- disease in an uniform population is given by the following deterministic equations, which are presented normalized with respect to the total population n . the groups s, e, i, r, h and d are the state variables of the dynamical system ( ). they are always nonnegative. the time derivativesṘ andḊ are also nonnegative, because recovered people and death cannot decrease, whereasṠ is always nonpositive, because we consider that recovered people cannot be reinfected. this fact is represented in fig. because the states r and d only have input arrows and the state s only has an output arrow. the model ( ) is a nonlinear system normalized with respect the population n , considered as a constant. hence s + e + i + h + r + d = and all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . the rate processes are modeled as follows. • αse and βsi are the transmission rates of the virus between the susceptible and the exposed population (respectively, infected population). α and β are the probability of disease transmission in a single contact with exposed (infected) people times the average daily number of contacts per person and have units of /day. typically, α is grater than β, assuming that people tend to avoid contact with subjects showing symptoms or diagnosed as positive. contacts between susceptible people and hospitalized people are neglected, excepting for healthcare workers. the probability of contagion from dead people is also neglected, despite the fact that some cases were recently reported. of course, recovered people are no longer able to transmit the virus. • u ∈ [ , ] is the effectiveness of nonpharmaceutical public health interventions (npis). u = means no intervention and the epidemic grows completely free, whereas u = implies total elimination of the disease spread. • ν is the vaccination rate, at which susceptible people became unable to be infected. unfortunately, in the covid- case ν = yet. • p is the probability that exposed people develop symptoms, γ − is the average period to develop symptoms, and ζ − is the average time to overcome the disease staying asymptomatic. • p is the probability that infected people with symptoms require hospitalization, δ − is the average time between infection and the need for hospitalization, and η − is the average time in that infected people recover without hospitalization. • p is the probability of hospitalized people die, − is the average time between the hospitalization and the death, and µ − is the average time to recover after hospitalization. the parameters used in ( ) are not very precisely determined and even differ greatly in the literature consulted (see [flng + , lgwsr , kan , lzg + , gbb + , jon , org] among many other references). most of the model adopted in the references adjust these parameters to match real data from different countries. it must be taken into account that some of these all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . parameters, mainly α and β, are not independent either from the populations and their general health status or their actions. the parameters α and β are related with the basic reproduction number r , defined as the expected number of secondary cases produced by a single (typical) infection in a completely susceptible population [jon ] . r is not a fixed number, depending as it does on such factors as the density of a community, the general health of its populace, or its medical infrastructure [shd ] . this is the most important parameter to understand the spread of an epidemic. if r > , the epidemic growths and the number of infected people increases. if r < , the epidemic decreases and after a certain time disappears, when a large enough number of people acquire antibodies and the so-called herd immunity occurs. in the actual covid- disease, r was determined to be . in wuhan, china [shd ] (between . and . according to [slx + ]), ranging from . to . in italy [shd ] and even close to . [lgwsr ] . an important remark is that many works consider r depending on the npis, admitting that these actions tend to reduce this number because the contact rates between people decrease. note that npis always occur even in countries where no government action has been taken, because people spontaneously tend to stay at home and to avoid contact with others. this fact explains the disparity of this number in different countries and reported in the references (see [lgwsr ] ). here, we consider r as a constant reproduction number in the absence of any external action, i.e., as if the disease could spread completely free, which, of course, is an unrealistic scenario. specifically, the relation between the rates α and β and r , can be calculated in model ( ) as in [jon ,gbb + ], resulting in section we propose a pair of values for parameters α and β to evaluate different epidemic scenarios. the effectiveness of the npis is considered in the variable u, which determines the rate at which susceptible people become exposed. several works [lzg + ,tbt + ,gv ,ssb ] consider these parameters as time dependent, because incorporate in these parameters the impact of governmental actions among other npis. the incubation period is estimated as γ − = . days [kan , flng + , twl + ]. the probability of developing symptoms p will be roughly estimated as all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . the period to overcome the disease without presenting symptoms is ζ − = . days (deduced from [gbb + ]). the infectious period with no need of hospitalization is widely accepted as days, so η = / . the probability to need hospitalization after the infection is p = % [low ,lzg + ,ssb ], and the time from symptom onset to hospitalization is δ − = . days [slx + ]. the probability to die after hospitalization is p = % according to [wm , flng + ] , and the average time to die is − = . days [slx + ]. the average time to recovery after hospitalization is µ − = days [flng + ]. finally, as it was noted above, there is no vaccine against this disease, so ν = . remark . of course, most of these parameters are subject to large inaccuracies, and they differ greatly in the literature consulted. however, as we will show below, the proposed control method is robust for such uncertainties as well as for measurements errors characterized as unreported or undiagnosed cases and inaccuracies in the group quantities. we propose the use of control theory to determine public nonpharmaceuticals interventions (npis) in order to control the evolution of the epidemic, avoiding the collapse of health care systems while minimizing harmful effects on the population and on the economy. as noted in [shd ] , "a properly designed feedback-based policy that takes into account both dynamics and uncertainty can deliver a stable result while keeping the hospitalization rate within a desired approximate range. furthermore, keeping the rate within such a range for a prolonged period allows a society to slowly and safely increase the percentage of people who have some sort of antibodies to the disease because they have either suffered it or they have been vaccinated, preferably the latter". the action law is given by the control variable u in ( ). no intervention from the public health agencies means u = , and the disease evolves naturally without control. at the other hand u = means the total impossibility of transmitting the virus, which, of course, is an unrealistic scenario. there are several possible choices of the reference signal or set point of the control system. one of them may be a small enough number of hospitalized people to not affect the capacity of the intensive care units (icus) available in the health care system. this reference signal maybe nonconstant, it can can go up because of an increase in available beds due to capacity additions in the health care system, by creation of provisory field hospitals, among other similar measures. by other hand, we must bear in mind that the quantities of each group described in ( ) are subject to large inaccuracies, due to unreported or undiagnosed cases, except for the number of people diagnosed as positive (i), which is quite well known, the number of hospitalized people (h) and, of course, the number of deaths (d). for that reason the output variable to be fed back only can be the infected population i or the hospitalized population h. hence, the goal of the control action is to keep the number of hospitalized people lower than the set point minimizing the external intervention which produces social discomfort and clearly harm the economy. therefore, the control action should aim to solve the following constrained optimization problem: where t is a considered period and sp is the reference signal (or set point, in the case it is considered as a constant). as a reference, the world health organization recommends a number of hospital beds per population, which means an index of . , or . %. this number will be used as the sp of the closed loop control system. however, we must bear in mind that npis impact on physical contacts between susceptible and infected or exposed people. if an individual is already infected, hospitalization will be required after at most δ − = . days or after δ − + γ − = . days on average if the infection was recent. hence, there exists a delay between the adoption of npis and their consequences on hospitalization of people. if the control action if calculated based only on the number of hospitalized people, the following . days too many people may require hospitalization, exceeding the capacity for medical care. in control jargoon, it means that there are almost two weeks with the system operating in an open loop. therefore, the control action needs to be calculated as a function of the number of infected people i (the number of exposed people e is quite unknown) in order to avoid future hospitalization requirements in the next . days at most. this strategy is known as predictive control. fig. shows the closed loop control system. the variable process is the infected population i and the control signal is the effectiveness of the npi all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. of course, in practical situations it is necessary to determine which actions and at what level correspond to a certain effectiveness of npis, but this issue is outside the scope of this paper. next, we show the results of different strategies of npis applied on the seihrd model. in this first series of experiments, we apply a constant control action u, that is, the system shown in fig. is an open loop control one. we consider as initial conditions i = e = . , h = r = d = , so s = . , that is, . % of the population is diagnosed as positive the first day and . % of the population is asymptomatic infected. during the first days of the epidemic, it was logical to consider that both exposed and infected people could spread the virus at the same ratio because the contagion between humans was not known. then, this disease could spread in a completely free scenario, in which no action is taken. this scenario has been called "naif " by several authors [ssb , lzg + ]. using the expression ( ) with r = . as in [ssb ,lzg + ], and assuming that no actions are taken during the epidemic, then α=β= . . the evolution of exposed, infected, hospitalized and dead people in this case is shown in the fig. . in this "naif " scenario, and using as initial condition infected and exposed person for different population values (n > , ) , the maximum are always . % for the exposed and . % for the infected, and the times when these maximums are reached depend on the population value n as is shown in the fig. . the delay between both maximums is a constant value all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. figure : population of exposed, infected, hospitalized and deaths group with no npi. naif scenario of days. additionally, the number of dead people forecasted by this model is about . % of the total population. clearly, this "naif " scenario seemed to be unrealistic since people tend to avoid contact with subjects showing symptoms or diagnosed as positive due to the severity of the covid- disease. in consequence, as we stated before, in a more realistic scenario α is greater than β. in the rest of this paper we consider β = α/ to take into account this assumption. fig. shows the areas of every group along the time in the case with no npi actions for illustrative purposes (with β = α/ ). fig. shows the evolution of the hospitalized with different constant npis effectiveness u and the proposed sp. table reports some results extracted from these simulations. the results presented in table show that, if no mitigation policy is all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . h (u= ) h (u= . *(t- )) h (u= . ) sp= . figure : population of hospitalized group with no npi (blue), with a npi of % effectiveness (yellow), with an intervention of % effectiveness applied weeks after the appearance of the first case (light blue) and sp (red). β = α/ adopted (u = ) approximately % of the population will be infected and . % will die. on the other hand, a relatively little aggressive npi, only % of effectiveness, is efficient in reducing the final number of deaths as well as the maximum number of hospitalized people, which is a crucial issue in order not to collapse the health system (the maximum value of h reaches the sp ). moreover, a late application of this strategy, after weeks since the first case arose, also significantly reduces these numbers. in this section, we simulate the behavior of the trajectories described by the normalized system ( ) subject to a proportional control action. the objective of the control action is that the number of hospitalized people does not exceed the number of available beds. of course, this number is highly variable in different countries, and can be increased during the duration of the epidemic with the construction of field hospitals, among other resources. on the other hand, as noted in sec. , to adopt as feedback variable the number of hospitalized people may lead to an overload of the health system in the following . days, for which a predictive control must be used that consider the number of infected people i. not all infected people need hospitalization. most of the symptomatic cases are mild and remain mild in severity [low , slx + ] ( − p = %). so we consider that p = % of infected people will need hospitalization in the following δ − = . days. this number plus the number of people already hospitalized h must remain below the set point. of course, we neglect the number of beds occupied by patients hospitalized for other diseases. hence, the proportional control variable is chosen as where k p is the proportional scalar gain with values between [ , ]. note that if i = , u = , and there is no need of a public intervention because no one is going to require hospitalization on the following . days, and with k p = , if a percentage of % of the infected people is equal to the number of available beds sp − h, u = which means that the public intervention must completely avoid the transmission of the virus because all these people will require hospitalization after δ − = . days on average. another point of view is to consider that this is a tracking trajectory problem, with a time dependent reference signal equal to r(t) = sp − h(t). we consider the same initial condition than in the former series of experiments, i = e = . , i.e. . % of the population infected and presenting symptoms at the first day and we suppose that . % of the population is infected and asymptomatic. fig. shows the trajectories of the variables vs. time with a gain k p = . note that the number of people hospitalized is always smaller than the set point. fig. shows the control signal vs. time. the control signal presents a maximum value of . , and the area under the curve of the control signal vs. time is . . of course, the smaller this action, the less the damage to the population and to the economy. the constant control signal equal to . presents an area under the curve equal to and equal to . when it is applied after weeks (see table ). we must bear in mind that npis are determined by governmental or popular decisions, and hardly can change every day as the control signal calculated by the proportional controller does. thus, we consider the application of npis with effectiveness shown in fig. . the amplitudes and times of this control signal were obtained from that shown in fig. . the detail of the trajectories of the states presented in fig. shows that there are no significant changes in the results presented. the maximum number of hospitalized people is . , the final number of deaths is . , and the area under the curve u vs. time is . . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . table shows the main results of the application of npis calculated using a proportional controller with different values of the scalar gain k p . in this section we consider the more realistic situation in which the parameters are partially unknown. as mentioned in sec. there are large uncertainties in the parameters, they diverge a lot according to the researched references and they are very different according to the country studied. in this series of experiments, the parameter α is randomly chosen is also randomly chosen between . and . . the parameter β between . and . . the incubation time γ − between and days.the probability to present symptoms p between % and %. the time of recovering between and days, for both symptomatic or asymptomatic people. the probability to be hospitalized p is considered as a gaussian distribution function of mean . and standard deviation of . . the time to be hospitalized δ − is chosen between and days. the probability to die p between % and %. the time to die − between and days. finally, the time of recovering from hospitalization µ − is randomly chosen between and days. in addition, we also consider that there exists some noncompliance of the nonpharmaceutical interventions. hence, we apply to the system ( ) a control signal with gaussian distribution of mean % of that calculated in ( ) with standard deviation of %, that is, we assume that there is % on average of noncompliance with the public measures adopted. the initial conditions are also i = e = . and the gain is k p = . fig. shows the trajectories of the states of the model ( ) during days since the first symptomatic case arose. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. figure : evolution of every group over time with a proportional control action with gain k p = (left) and considering % of noncompliance of the npis policies on average. the picture at the right is a zoom of that at the left. set point equal to . . table reports some results extracted from this series of simulations. the similarity of the results reported in tables and , as well as the trajectories shown in figs. and , show that the proportional controller is robust to parameter uncertainties and to some noncomplaince of the npis, which, of course, always occurs in practice. the proportional controller proposed to guide the adoption of npis shown its efficiency to keep the number of hospitalized people below a set point given by the health system capacity. moreover, this very simple strategy is robust to parameters uncertainties and to some level of noncompliance of the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . figure : control signal over time using a proportional controller and considering % of noncompliance of the npis policies on average. the blue curve is that calculated in ( ), the red curve is the control signal considering the randomic noncompliance. public measures. the control signal calculated by this method aims to guide the adoption of npis in order of minimizing the social impact and the economical damages. as an example, recently the argentine government relaxed some restrictions adopted in the quarantine period, allowing more economic and recreational activities in some cities. the only criterion used to adopt this measure was the number of days in which the number of infected people doubled (the so-called doubling time). even thought this decision also can be considered as a closed loop control action, the criterion adopted is a little improvised. an open question is how to translate the rate of effectiveness of the npi calculated by the controller into concrete actions adopted by governments or public health authorities. moreover, we must bear in mind that these measures cannot be continuously varied along the time, as the control signal does, but they are decisions that will remain valid for at least few days. however, although this issue is out of the scope of this paper, some decision can be changed every day, for example, the number of individuals with permission to leave their homes or the number of people allowed to get into a store, among other little decisions that can change every day according to the control variable suggests. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint simcovid: an open-source simulation program for the covid- outbreak impact of nonpharmaceutical interventions (npis) to reduce covid mortality and healthcare demand modelling the covid- epidemic and implementation of population-wide interventions in italy análisis del covid- por medio de un modelo seir notes on r modeling and control of a campus covid- outbreak annelies wilder-smith, and joacim rockl ov. the reproductive number of covid- is higher compared to sars coronavirus coronavirus: what are asymptomatic and mild covid- ? a conceptual model for the coronavirus disease (covid- ) outbreak in wuhan, china with individual reaction and governmental action estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship global health -seir model world health organization. coronavirus disease (covid- ) how control theory can help us control covid- the novel coronavirus, -ncov, is highly contagious and more infectious than initially estimated a mathematical description of the dynamic of coronavirus disease (covid- ): a case study of brazil an updated estimation of the risk of transmission of the novel coronavirus ( -ncov) effect of changing case definitions for covid- on the epidemic curve and transmission parameters in mainland china: a modelling study covid coronavirus pandemic key: cord- - x wklr authors: jiang, shanhe; zhang, dawei; irwin, darrell d. title: semiformal organizations and control during the covid- crisis in china date: - - journal: asian j criminol doi: . /s - - -z sha: doc_id: cord_uid: x wklr scholars often compare china and western societies in terms of dichotomous forms of control—formal and informal. recent research indicates a nuanced form of social control—semiformal. using a survey data collected during the covid- pandemic in china, this study investigated the prevalence and importance of semiformal organizations, formal organizations, and informal groups participating in social control and social service and the predictors of the perceived importance of these three forms of social control mechanisms. findings from this study revealed that formal organizations, the semiformal organizations, and informal groups all participated in community control and service. semiformal organizations had the highest level of participation. this study found that education and age are the two major predictors of the views on the importance of formal, informal, and semiformal control and control mechanisms. order. on january , hubei invoked a class response to public health emergency (han ) . under the lockdown order and class i response, the city's public transportation services including bus, metro, and ferry services were suspended. the city's airport and railway stations were also suspended except for exceptional circumstances. residents were first advised and then ordered to shelter in place and only allowed to leave their residence for essential activities (china cdc ; hubei.gov ) . during the period of lockdown, the local community played an essential role in food supplies, health screening, collecting and distributing the covid- -related information, supplying personal protective gear, setting up checkpoints to monitor people who enter and exit communities, made drug purchases for those who faced difficulties obtaining medicines on their own, and carried out duties of community sanitization. other major cities and provinces quickly followed suit to launch a lockdown or issue a class response to public health emergency. by january , all provinces (or equivalents) in mainland china had launched a class response (caixin ) and followed similar measures as wuhan and the other cities in hubei province. starting on march , cities in hubei province, except for wuhan, rescinded strict travel restrictions, and the citizens' daily lives began to gradually return to normal. on april , wuhan lifted its lockdown, being the last city in hubei and china to remove such strict controls (mao et al. ). traditional classification of social control is dichotomous: formal and informal (black (black , rose and clear ) . formal social control is a control imposed by law and implemented by government or government agencies such as courts, police, and prisons, while informal social control is a control carried out by informal groups such as neighborhood watches and kin mediation based on moral rules (huang a; jiang et al. ; liu et al. ; rosenbaum ) . however, when this dichotomous classification of social control is applied to china, it overlooks an important form of social control-semiformal social control (huang a (huang , b (huang , jiang ; jiang et al. ; jiang et al. ) . during the early period of china's economic reform, researcher ronald troyer visited china five times from to to observe the chinese responses to crime and deviant behavior. his visitations, observations, and conversations with chinese officials and citizens, along with his examination of news media and academic literature review impressed upon him that two grassroots organizations-the residents' committee and the residents' groups-played an important role in social and crime control. he noted, however, that "they are not voluntary association in the u.s. sense; they are government organized and controlled entities" (troyer : p. ) . although troyer did not use the terms such as semiformal governance or control, he implied that these grassroots organizations are different from those informal groups and informal control in the usa. in their article published in , zhang and his associates examined the importance of tiao-jie and bang-jiao in social control and crime prevention in china. according to xiong ( ) , tiao-jie is a form of mediating disputes that is carried out by the people's mediation committee, which is under the leadership of the residents' committee in urban areas and of the village committee in rural areas. another form, "bang-jiao means assisting, helping, guiding, and directing offenders, especially juvenile offenders" (zhang et al. : p. ). both tiaojie and bang-jiao groups are neither completely formal nor informal control devices. they are mass-roots organizations, both imposed and heavily influenced by the government, with rules for social behavior. chen ( ) compared and contrasted chinese and western social and legal control. he found that "some informal institutions such as neighborhood committees do not form a part of the formal criminal justice system, yet they are established based on certain legal regulations and often are trained by local justice organs or police" (chen : p. ). thus, informal groups or organizations such as neighborhood committees in china are "quasi-formal." huang ( ) reported similar forms of social control and control mechanism. using his archival research of documents from different periods of history in china, huang ( ) revealed a form of governance in dispute resolution which differs from that in the west; he called it semiformal governance. through his archival research of the qing dynasty's ( - ) approach to civil justice or minor matters, huang ( ) found that many small disputes such as arguments over land, debt, inheritance, and marriage were usually resolved by interventions by the local community or kin mediation. for minor matters, only when disputes could not be resolved by the local community, the court got involved. xiangbao (乡保) played an important role in handling local issues and was the liaison between the court and the community mediation. xiangbao was an unsalaried quasi-official recommended by the local community and confirmed by the state. the court heavily relied on xiangbao's work for its decisions on dispute cases and preferred to use informal and semiformal processes to resolve them. the widespread use of an unsalaried quasi-official in the process of disputes is semiformal governance. huang ( ) also found that the semiformal governance in the late qing not only existed in civil justice but also was frequently utilized in tax administration and educational administration. in fact, semiformal governance continued to exist in the republic period and the people's republic of china ( -present) of today. in their test of the generalizability of social disorganization theory, jiang et al. ( ) also used social and crime control carried out by residents' committees as semiformal control and examined its impact on crime. based on the survey data, they revealed that semiformal control did decrease the perceived neighborhood property crime in guangzhou, china. in , jiang reviewed the conceptual and empirical articles which pertain to the forms of social and crime control and stated that semiformal control is one of the chinese characteristics in social and crime control (jiang ) . in , jiang and his associates expanded the review of the forms of social and crime control and concurred with huang ( ) that traditional research of the forms of formal and informal social and crime control overlooked an important form of control-semiformal. semiformal control is the integration of formal and informal controls (huang a (huang , b (huang , jiang et al. ) . semiformal control positions features underlying formal control and informal control but uniquely has features neither found in informal nor formal control. control carried out by semiformal organizations is the core part of semiformal control. according to jiang et al. ( ) , "semiformal organizations integrate formal and informal organizational features; they are grassroots organizations but are marked by the government; they are nongovernmental organizations but have official power and responsibilities; they are a bridge between the state and society." (p. ). jiang et al.'s study ( ) not only described the semiformal control features of chinese community corrections but also empirically compared and contrasted correctional staff workers (representing formal control) and community committee members (representing semiformal control) in terms of individual characteristics, views of the criminal justice system, and the supervision strategies. encompassing a number of direct observations and studies, jiang et al. ( ) summarized three forms of social control: formal, informal, and semiformal. formal social control is a "control imposed by law and/or implemented by official controlling organizations" (jiang et al. , p. ) . policing and corrections are typical examples of formal social and crime control. informal social control is a "control carried out by unofficial controlling groups based on moral rules" (jiang et al. , p. ) . shaming by family members and neighbors is an example of informal social control. collective efficacy proposed by sampson et al. ( ) is another example of informal social control acting to mediate social problems. semiformal social control is the mix of formal and informal controls (huang a (huang , b (huang , . based on both controlling actor and rules of control, jiang et al. ( ) defined semiformal control as controlling action that is carried out by ( ) semiformal organizations based on sentiment, reason, and law, and ( ) government agencies or other formal organizations based on sentiment and reason. although government agencies and other formal organizations may use sentiment and reason to help their controlling action, their primary or basic action rules are laws, policies, and formal regulations. in addition, using jiang et al.'s ( ) definition would be difficult to classify or decide whether government agencies as formal or semiformal. thus, the current study uses controlling actors as a criterion and defines semiformal control as controlling action that is implemented by semiformal organizations. semiformal social control can be manifested in diverse ways in china. for example, when a community corrections group, consisted of local justice officers, community committee members, volunteers, and family members, employs the law and regulations to re-educate and reform an offender, semiformal control occurs (jiang et al. ; jiang et al. ) . when the mediation team within a community or a grid, which is usually composed of the communist party organization building, resident's committee administration, and residential volunteers, resolves disputes between residents based on laws and local norms (tang ) , semiformal control occurs. more broadly speaking, when social or crime control is carried out by semiformal organizations, semiformal social control occurs (jiang et al. ; jiang et al. ). communities (shequ) in china are large and widespread semiformal organizations. every local residential community is administered by a resident's committee in urban areas while it is managed by a village committee in rural areas (jiang et al. ) . accordingly, the last type of semiformal control is the most commonly used in china's social and crime control (jiang et al. ) . thus, a body of literature has emerged regarding the important role that semiformal organizations play in china's social control and social services. important semiformal organizations such as residents' committees and village committees bridge government and individual citizens. the government provides services to citizens and supervises citizens through community committees. semiformal organizations and controls make it possible for the chinese government to effectively deal with emergencies or crises such as the covid- outbreak. however, there is limited quantitative research on the investigation of the prevalence and importance of semiformal organizations in social control and service. this study was undertaken as an academic project to glean data on the use of social control during the covid- pandemic. the study also intended to quantitatively explore what stated and unstated factors explain the perceived importance of control. this research formulates the first effort to search for the predictors of the importance views. since there were no clear theoretical guidelines for the predictor selection, the previous literature on the importance of social control forms were used for the variable selection employed in this study. he variables gender, age, and education are often included as demographic characteristics in the extant studies (jiang et al. a; jiang et al. b; jiang et al. ; jiang et al. ; lambert et al. a; lambert et al. b) . following suit, this study also included them. the study additionally included another characteristic-political affiliation-whether the respondent is a communist party member. this variable is essential since the party membership is expected to be in favor of formal control mechanisms such as government agencies/officers and the police. living area is another commonly examined correlate of the views of forms of social and crime control jiang and lambert ; jiang et al. ; jiang et al. ) . since the outbreak of covid- in wuhan, the virus subsequently spread across china and around the world at different points. thus, this study also included two dummy variables (wuhan; hubei) to examine whether wuhan and hubei differed from other areas in china in the views of three forms of social control and social service. as for gender, in the west, particularly in the usa, women are encouraged to think independently. due to different socializations, especially during childhood, women are more likely to use a "care voice" reasoning for their response to many social and criminal justice issues, while men are more likely to use a "justice voice" reasoning to express their opinions (gilligan ) . women are often linked to an empathic, caring attitude toward criminal justice issues, while men are more likely to be impartial and justice oriented ). in china, women occupy different cultural, political, and social environments. on the one hand, when china progressively becomes modernized, women receive more education and become more economically independent, and they are encouraged to think more independently. on the other hand, patriarchal tradition, collectivism, and the one-party political system all encourage, promote, or even pressure different genders to think similarly . it is uncertain whether gender affects the views of different forms of social control and social service. in the past studies about the views of formal and informal control in china, results are mixed. most studies did not find an association between gender and the views of forms of social control. when lambert et al. ( ) examined whether gender differed in statements (some of them measured formal or informal control), gender did show differences in some statements but not for others. jiang et al. ( a) reported men were more likely than women to believe that formal control such as police, courts, and incarceration is deterrent to crime. however, the sample used in jiang, lambert, and jenkins' analysis included respondents from china and the usa. clearly, more research on gender and the views of forms of social control is warranted. age is commonly believed to be positively associated with the views of social control and social service. in other words, younger people are less likely to value social control than their older counterparts are. two possible reasons behind this assumption are that younger people are freer from constraints, and younger people are more likely to self-support and be self-sufficient. three studies quantitatively examined whether age is associated with the views of informal and formal social control in china. two studies jiang et al. b) revealed no relationship existed between them. one (jiang et al. a ) reported a positive relationship. when a specific formal agency was evaluated, age sometimes was found to be related to the views of formal control or formal control organizations. for example, sun et al. ( ) found that age was positively related to perceived police legitimacy and cooperation with police. inconsistent findings in the previous studies invite more research on age as a variable. education is also used to predict the views of formal and informal social control but in the lesser degree, relative to gender and age. generally, education leads to more critical and independent thinking that in turn results in the less favorable assessment of the importance of formal organizations in social control and more favorable attitudes toward the importance of informal groups in social control. two empirical studies reported mixed results concerning informal social control. one study conducted by jiang et al. ( b) found that education was not associated with the perceived importance of informal control in neighborhoods in guangzhou, china. another study conducted in hubei by jiang and lambert ( ) reported that more educated people perceived more importance of informal groups in social control but perceived less importance of formal organizations. during the covid- crisis, governments in each nation have played a leading role in social control and social services. at the community level, the importance of government in social control and social services may vary from society to society. political affiliation could affect the perceived importance of government and other control mechanisms in china. furthermore, chinese communist party members may have more favorable ranking of the importance of formal control mechanisms such as government agencies and police than non-party members may. in contrast, non-party members may believe that semiformal organizations such as residents' committee/village committee and volunteers are more important in social control and service at the community level. thus, this study included party affiliation to examine whether the party membership affects respondents' views of forms of social control and service mechanisms. people who grow up in rural areas are socialized differently from those who grow up in urban areas. the cultures of rural areas have more traditional values with more personal interactions and thus are more cohesive than the culture of large urban areas. people who grow up in rural areas are more likely to use and believe in informal mechanisms such as family, neighborhood, and peers in maintaining community order and providing or obtaining mutual support. jiang et al. ( ) found that compared to people from urban areas, people from rural areas were more likely to rank the importance of informal control in crime prevention higher and the importance of formal control lower in china. however, jiang and lambert ( ) did not find the connection between living areas and ranking of the importance of formal and informal mechanisms in social control or crime prevention. this study further examined whether urbanization affects people's views of the importance of forms of control mechanisms in social control and services. our research anticipated that, relative to respondents from urban areas, respondents from rural areas rank the importance of community organizations and informal groups higher in terms of social control and service. for the importance of formal organizations in social control and service, the urbanization impact may be murkier. people from rural areas have more traditional values and may have high trust and confidence in government (irwin et al. ) . they may be more likely or equally likely to value the importance of government agencies in social control and service relative to their urban counterparts. the last two variables included in this study are based on the unique situation of the covid- crisis. covid- was first reported in wuhan and then spread throughout china and the world. wuhan experienced the most severe public health crisis in china. accordingly, social control and service needs in wuhan and surrounding areas in hubei may also differ. people from different areas may also view the role of government agencies, police, community, volunteers, and regular citizens in maintaining community order and providing service differently. thus, this study created two dummy variables to see whether people from wuhan, hubei, outside wuhan, and other provinces have different views of the importance of three forms of social control. based on the reported covid- cases in china, there is clear ranking order of severity of the outbreak among these three areas: wuhan, the capital of hubei province; hubei excluding wuhan; and other provinces. the research team planned to collect data from approximately people from each of the three regions. semiformal control and semiformal organizations are prevalent and play an important role in china's governance including social control and social service (jiang et al. ) . to quantitatively verify the prevalence and the importance of semiformal control and semiformal organizations, the research team in china designed the survey instrument and conducted the surveys. the questionnaire used in the survey was designed based on the semiformal control theory proposed by jiang et al. ( ) and additional literature on formal and informal control. the first questionnaire draft was completed in march and then pretested in hubei and eight other provinces at the end of march. in the early april, the revised questionnaire was pretested again in hubei including wuhan. considering memory will be gradually lost with time, the survey organizer in china recruited interviewers from wuhan, hubei, and other regions to speed up the pace of the surveys. these interviewers were a mixture of current graduate students or graduates from the organizer's school and had experience in conducting previous surveys. all the interviewers were trained on how to conduct this survey in early april. the survey began on april and ended on april , . during this period, although china lifted its lockdown orders in all areas except in wuhan, many places still had restrictions on travel or person to person interactions. thus, to collect valid data and improve the response rate, two data collection methods were employed. one method was to send the survey instrument to the interviewees via the chinese social media wechat app or other digital portals where respondents were to answer the questions by themselves. using this method, the interviewers were required to be available to answer any questions when a respondent was completing the questionnaire. doing so aimed at helping improve the completion rate for the entire survey instrument as well as each individual question. the second survey method used to collect data was via an online platform such as zoom or wechat or in face-to-face interviews. using this method, the interviewers asked questions and recorded the interviewees' responses. all the respondents were informed ( ) who the survey team were; ( ) the survey participation was voluntary; and ( ) the data from the survey would be confidential. through this process, respondents were assured if the collected data are used for publications or reports, no individual respondents would be identified. seven hundred questionnaires (online or in print) were distributed, and valid questionnaires were used in this study. successful response rate was slightly more than %. one of the survey's purposes was to collect data on the prevalence of formal, informal, and semiformal control or organizations in maintaining community order and providing service during the covid- lockdown. government agencies or officials and community police or police officers are the indicators of formal control mechanisms. volunteers and regular citizens are the indicators of informal control groups or individuals. community committees or committee members are the indicators of semiformal control mechanisms. thus, questions pertaining to the prevalence of three forms of control ask whether each of these actors participated in community control and service during the lockdown period. related to the prevalence, the respondents were also asked to judge how important each of these organizations or their representatives was in six essential community control and service activities during the covid- lockdown: checkpoint inspection, health screening, food supplies, drug supplies, sanitization, and information distribution. importance here refers to an appraisal of the level of involvement of a given actor. the response categories for these questions were not important at all (= ), not important (= ), between not important and important (= ), important (= ), and very important (= ). the score summation of these six activities formed the five dependent variables for five-control-service mechanisms: community, government, police, volunteer, and citizen. independent variables in this study refer to those predictors of the views of forms of community-control-service mechanisms. they include age, sex, education, party, locationurban or rural-and two dummy variables for region: hubei and wuhan; the reference group is other regions. age was measured in years. sex was measured by male (= ) and female (= ). education was measured by the highest degree completed or was studying for at the interview time. the answer categories were elementary or less (= ), junior high (= ), high school (= ), technical school (= ), -year college (= ), bachelor's ( -year) degree (= ), and graduate school (= ). party was measured by whether a respondent was the communist party member with yes = and no = . location was measured by whether a respondent came from an urban area or rural area. the two dummy variables are hubei (= ) and wuhan (= ). table is constructed to meet the first goal of this study: investigate the prevalence of semiformal organizations participating in social control and service during the covid- lockdown. the table is based on the data from the respondents' answers to this question: "during the covid- lockdown period, who participated in the following activities: checkpoint inspection, health screening, food supplies, drug supplies, sanitization, and information distribution." the choices included residents' or village committee, government agencies or officers, community police or police officers, volunteers, regular citizens, and no action. the respondents could make multiple choices for each activity item. in this survey, volunteers were those who registered with a residents' committee in urban areas or a village committee in rural areas to volunteer their time to help the community control and service activities. regular citizens refer to any citizens who voluntarily helped the community control and service activities but did not register with the community. visual inspection of table revealed several points. first, relative to other activity items, demand for food and drug supplies via community was lower. this finding makes sense because only a small portion of people need drugs. in addition, citizens who were not infected by the virus or their neighbors in a same multi-story building who were not infected by the virus were usually allowed to go out to buy life necessities by themselves, though they were limited to one person per family at certain time periods. second, community (resident's or village) committee had the highest participation in all the activities. this finding is not surprising. according to china's residents' committee act of , a community is a self-governing unit. each community has a residents' or village committee, in which members are local citizens and elected by local citizens and approved by the government (jiang et al. b) . the committee has many neighborhood functions, including but not limited to "distributing and promoting laws and policies from the central government and local government, settling disputes, assisting the local police department to maintain public order, …, household registration, local sanitation and cleanliness activities, encouraging residents to practice birth control, and a variety of other services to the local residents, such as establishing senior centers" (jiang et al. b, p. ) . during the covid- lockdown, the residents'/village committee was instructed by the government to participate in the activities listed in table . third, overall, volunteers had the second highest participation in the table activities. china is a collective society, and things or activities are usually organized and done by the local community and government. however, during these lockdown times, many people volunteered their time, resources, and vehicles to help needy people in the community. this voluntary outpouring was a particularly good thing happening in this challenging time. table showed that volunteers actively participated in all activities except distributing the healthrelated information. a relatively low level of participation in the activity is understandable because this information is more likely from the government. the government mobilized to address the covid- health crisis at multiple levels, most prominently flying in thousands of nurses and doctors from around china to wuhan and other hubei cities (xinhua news ). fourth and the last, the participation of community police officers in table activities was not high. there were at least two reasons behind the low level of the participation. first, a community usually has one officer so their participation was not easily observed. second, the officer's major role is to assist the community committee to maintain community order. during the lockdown, outdoor activities were reduced to a minimum, and as a result, deviant or criminal behavior was also greatly decreased. thus, the officer's social control action was also lessened. in sum, formal organizations, government agencies and police, the semiformal organizations, residents' committee in urban areas and village committee in rural areas, and informal groups or individuals, volunteers, and regular citizens, all participated in community control and service. semiformal organizations had the highest level of participation during the lockdown period. table is constructed to meet the second purpose of the study: investigate the importance of semiformal organizations in social control and service, compared to formal organizations and informal groups. the table presents the perceived importance of five types of actors in the six activities. consistent with the level of participation in the activities, the importance of the community committee was the highest while that of regular citizens was the lowest. in addition, community police officers were ranked relatively high in mandatory controlling items such as a checkpoint inspection and health screening and low in-service items such as helping shop for necessities and medicine. next, the importance of government agencies or officers was relatively low in shopping for necessities and medicine and relatively high in other items such as health information distribution and health screening. this finding makes sense because the governmental role at the community level was more focused on directing and supervising action implementation rather than specific action. although the government sent one or more officials to each community, their visibility to citizens was low. in contrast with government agencies or officers, the importance of volunteers in shopping for necessities and medicine was very high. their high participation or action in these activities may be the reason behind this. examining table , government services acted as the superstructure providing guidance with the volunteers acted informally to meet daily needs. the findings from table confirmed the importance of semiformal organizations in maintaining community order and providing community service during the lockdown period. the reported importance of semiformal organizations is higher than that of formal organizations and informal groups. tables , , and meet the third purpose of the study: discover the predictors of the views of forms of social control and service. table presents the descriptive statistics of the dependent and independent variables in this study. the mean score for each dependent variable in the table was created from the simple summation of scores in the six community activity items. as revealed in table , the summarized mean scores in table confirmed that community had the highest score, government the second, volunteer the third, police the fourth, and citizen the last. for the independent variables, about % of the respondents were members of the chinese communist party. almost % of the respondents were from rural areas while the other % from urban areas. more females ( %) participated in the survey. the mean age of the respondents was approximately . most respondents had a bachelor's degree or were studying at a -year college. table presents the results from bivariate regression analyses of all independent and dependent variables. table shows the findings from multiple regression analyses of the each of the actors in this table was ranked in terms of their importance in the six activities. the answer categories concerning the importance are not important at all (= ), not important (= ), between not important and important (= ), important (= ), and very important (= ) effect of all the independent variables on each dependent variable. several significant relationships in table disappeared in table . visual inspection of tables and shows that education is the strongest predictor of the views of all the five control and service mechanisms: communities, governments, police, volunteers, and regular citizens. it is the only predictor that is statistically significant for all the five control and service mechanisms. one possible reason behind the finding is that people who are more educated are more critical and have higher expectations about the role each control and service mechanism should play. thus, relative to those with lower education, the respondents with higher education were more likely to give control and service mechanisms lower rankings of their level of involvement in control and service activities. next, age is a significant predictor for the views of government agencies/officials and community policing officers. furthermore, younger people had more favorable views. this is surprising. the findings are neither consistent with the expectations in this study nor the ( ) the two dummy variables hubei and wuhan were entered into the regression model together. ( ) results from independent t tests for comparing two means showed the same patterns as in this table regarding whether a relationship between two variables (one nominal independent variable with importance view scores) is statistically significant *p < . , **p < . , *** p < . findings in the previous studies of formal and informal control jiang et al. b; jiang et al. a) . do these findings suggest that younger people have more trust and confidence in government in community control and service? a further examination reveals that the mean age of the respondents is about . many of them have grown up at the time when china's economy developed quickly and has improving international visibility. relative to older people, they may be fortunate with no experiences of economic hardship in their life. they may be still in school and have been educated to hold a positive view of the government and china. acknowledging different lived experiences, it is worth further examining the linkages between respondents' ages and views of control mechanisms and support for government policies. another surprising finding from table is that party membership was negatively associated with the importance views of government and police. that is, the communist party members ranked the importance of government and police in maintaining community order and providing community service during the lockdown lower than non-party members did. in order to explore the reason behind these unexpected findings, this study used the forward selection method to control other variables in multiple regression analyses or multi-factor anova tests. results (not shown here but available upon to request) reveal that when education was controlled for, the relationships between party and the views of government and the views of police disappeared. these findings suggest that party members had less favorable views on the importance of government and police because they were more educated. the educated party members may recognize that disasters tend to disrupt bureaucratic goals and tasks when predictable social harmony and stable growth have been put forward as party goals to the public (irwin and willis, ) . table also shows that respondents from urban areas ranked the importance of government and regular citizens in community control and service lower than those from rural areas. a further examination (results not shown here but available upon to request) reveals that when education was controlled for, city dwelling became statistically insignificant for government, and when both education and age were controlled for, city dwelling was no longer a predictor of the views of regular citizens. a further examination also showed that education and urban life intervened in the relationship between wuhan and volunteers and partially explains the relationship between hubei province and volunteers. furthermore, when the variables education and city were controlled for, the relationship between wuhan and volunteers disappeared, findings reported in the last section are constrained; first, the sample used in this study was not randomly selected and findings cannot be generalized to the larger population with a known error, and second, due to the covid- pandemic, people in china had limited interpersonal interactions during the survey time period. the online questionnaire survey and interviews were important methods to collect this purposive data while the response to covid- was still ongoing. internet access and a certain education level were required to participate in the surveys/interviews. as a result, the respondents surveyed in this study are younger and more educated than the general population. in addition, the proportion of the chinese communist party members ( %) in this study is higher than that ( %) of the general population aging and older, suggesting that party members, not intentionally, were oversampled. these sampling biases can result in the over-or underestimation of the corresponding population values. in this regard, the nature of this study is exploratory. findings from this study are preliminary rather than conclusive. nonetheless, this study contributes to the research on forms of social control and social governance in several ways. this is the first to quantitatively investigate the prevalence of formal, informal, and semiformal control and control mechanisms simultaneously. thus, the study was able to provide us with comparative results of the participation level of each organization or group and their levels of control employed in maintaining community order and providing community service during the unique covid- lockdown conceived in china. results from this study provide complementary evidence that semiformal organizations and control are prevalent in china's social control and governance (jiang et al. ). this study on the covid- response in china is also the first to quantitatively evaluate the importance of formal, informal, and semiformal control and control mechanisms in maintaining community order and providing community service. the findings regarding the conceived importance allow us to not only compare the relative importance of each form of social control and service in china but also eventually find commonalities and differences with other nations in terms of covid- control mechanism using the survey methods described herein. the findings regarding the conceived importance also help us understand why semiformal organizations such as residents' committee and village committee play such a prevalent part in china's social control and services. finally, this study found that education and age are the two major predictors of the views on the importance of formal, informal, and semiformal control and control mechanisms. the findings confirmed the expectation that more educated people were more critical and expected more from all forms of control mechanisms in social control and service so they ranked the importance of all forms of control mechanisms lower than less educated people. findings from this study are inconsistent with our expectation that older people are more likely to have a favorable ranking of the importance of government agencies/officials and police in social control and service. some possible reasons are explored in the "findings" section, but more research is required to examine the age-importance view linkage. with a current focus fixed on the covid- pandemic, these findings from citizen respondents in china, though an important contribution, are preliminary, and more relevant research is needed in the future. the behavior of law 中国内地 省份全部启动突发公共卫生事件一级响应 (thirty one provinces or equivalents in mainland china lunched a class i to public health emergency social and legal control in china: a comparative perspective notice of tightening the prevention and control covid- crime and crime prevention in china: a challenge to the development-crime nexus in a different voice: psychological theory and women's development 武汉时间:从专家组抵达到封城的谜之 天 (wuhan days between informal mediation and formal adjudication: the third realm of qing civil justice public sphere"/"civil society" in china? the third realm between state and society centralized minimalism: semiformal governance by quasi officials and dispute resolution in china chinese civil justice, past and present notice of further tightening restrictions amid the covid- crisis success or sorrow: the paradoxical view of crime control campaigns in china china's social transformation and the development of rural community corrections social and crime control with chinese characteristics views of formal and informal crime control and their correlates in china correlates of formal and informal social/crime control in china east meets west: an empirical comparison of formal and informal crime control among chinese and the u.s. students correlates of informal social control in guangzhou, china neighborhoods university students' views of formal and informal control in japan: an exploratory study formal and informal control inchina social ties, collective efficacy and perceived neighborhood property crime in guangzhou semiformal crime control and semiformal organizations in china: an empirical demonstration from chinese community corrections semiformal control: a paradigm from chinese society a preliminary study of gender differences on views of crime and punishment among chinese college students correlates of formal and informal social control on crime prevention: an exploratory study among university students formal and informal crime control views in bangladesh and the united states crime and social control in a changing china 武汉解封 (wuhan lifted the lockdown order chinese social control: from shaming and reintegration to "getting rich is glorious incarceration, social capital, and crime: implications for social disorganization theory community crime prevention: a review and synthesis of the literature neighborhoods and violent crime: a multilevel study of collective efficacy procedural justice, legitimacy, and public cooperation with police: does western wisdom hold in china? grid governance in china's urban middle-class neighborhoods social control in the people's republic of china how does china combat #coronavirus: more than , medics across the country came to wuhan criminal justice systems in china crime prevention in a communitarian society: bang-jiao and tiao-jie in the people's republic of china bicycle-theft victimization in contemporary urban china: a multilevel assessment of risk and protective factors publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. informed consent yes key: cord- - zxgaprl authors: asamoah, joshua kiddy k.; owusu, m.a.; jin, zhen; oduro, f.t.; abidemi, afeez; gyasi, esther opoku title: global stability and cost-effectiveness analysis of covid- considering the impact of the environment:using data from ghana date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: zxgaprl covid- potentially threatens the lives and livelihood of people all over the world. the disease is presently a major health concern in ghana and the rest of the world. although, human to human transmission dynamics has been established, not much research is done on the dynamics of the virus in the environment and the role human play by releasing the virus into the environment. therefore, investigating the human-environment-human by use of mathematical analysis and optimal control theory is relatively necessary. the dynamics of covid- for this study is segregated into compartments as: susceptible (s), exposed (e), asymptomatic (a), symptomatic (i), recovered (r) and the virus in the environment/surfaces (v). the basic reproduction number [formula: see text] without controls is computed. the application of lyapunov’s function is used to analyse the global stability of the proposed model. we fit the model to real data from ghana in the time window th march to th may , with the aid of python programming language using the least-squares method. the average basic reproduction number without controls, [formula: see text] is approximately . . an optimal control is formulated based on the sensitivity analysis. numerical simulation of the model is also done to verify the analytic results. the admissible control set such as: effective testing and quarantine when boarders are opened, the usage of masks and face shields through media education, cleaning of surfaces with home-based detergents, practising proper cough etiquette and fumigating commercial areas; health centers is simulated in matlab. we used forward-backward sweep runge-kutta scheme which gave interesting results in the main text, for example, the cost-effectiveness analysis shows that, strategy (cleaning of surfaces with home-based detergents) is the most cost-effective strategy among all the six control intervention strategies under consideration. that other optimal control model on covid- have been studied (see for example [ , , , , , the model further assumes that, no exposed individual transmits the disease. the proportion of those in e class into both a and i classes is given as k ( − γ)e and k γe respectively. similarly, individuals recovering from the a class is v ( − φ)a and those joining the i class from the a compartment is v φa. though, not much had been said with regards to the possibility of the recovered individuals joining the susceptible population. all the same we included it so to ascertain the impact short and long-term immunity on the dynamics of covid- , denoted as ρ, where ρ ≥ . table progression rate from exposed to the symptomatic (severely infected) class k progression rate from exposed to the asymptomatic class given that s ≥ , e ≥ , a ≥ , i ≥ , r ≥ and v ≥ . we simplify equation ( ) to get the total differential equation as; where n = s + e + a + i + r. non-negative such that the initial conditions are also given as the proof of theorem can be obtained using the procedures presented in [ ] , as shown below. proof. we let y = (s, e, a, i, r, v ) t and k = βi, k = βa, k = β v where t is transposition, then our differential equation ( ) can be rewritten in a matrix form as dy now, using the third equation in model ( ), thus and deploying the method of integration factor and change of variables [ ] , yields t e(s)e −(ω+v φ+v ( −φ))s ds . next, we consider the fourth, fifth, sixth equation of model ( ) and using the same above process, we have written as where m = (m + m ). proof. from the first equation of ( ) we have now, take m to be a solution which is unique to the initial value problem which when solved gives hence, by the comparison principle (see for instant theorem of [ ]), it accompanies that also from the second equation of ( ), we let m = (m + m ) with the assumption that < a + i ≤ Λ ω . then, now, let m to be a solution which is unique to the initial value problem which when solved gives and, by the comparison principle (see for instant theorem of [ ]), it accompanies that from ( ) and ( ) here, we first focus on equilibrium points when there is no disease in the system. considering equation ( ), we put e = a = i = r = v = . this indicates that, there is no disease in the system at this stage. therefore, solving for the stationary points, we have e = (s * , , , , , ) where s * = Λ ω . for the r , we use the concept of the next generation approach. here, we seek to find the average number of new infections given that an infected individual is introduced into the population under study [ ] . we let g be the next generation matrix which consists of is the rate at which a new infection occurs in compartment i. also, v + i and v − i are the rate of immigration into compartment i and the rate at which new individuals are transferred from compartment i respectively. we note that, all the functions are continuously differentiable at least twice [ ] . now the next generation matrix is defined as; hence, the r is given as the maximum absolute eigenvalue of the next generation matrix (g) given that, σ contains all the eigenvalues of g. this eigenvalue is known as the spectral radius (ρ). this is represented as among the infected classes (e, a, i, v), we have f i as finding the jacobian of the matrix f i gives considering the same compartments (e, a, i, v), we get the matrix v as finding the jacobian matrix of v i gives after computing for the eigenvalues of the matrix g, we have that the maximum absolute eigenvalue, r is given as; secondary infection seeded by i state through direct contact secondary infection seeded by i state through the environment ( ) which can be written as where r a is the secondary infections generated by asymptomatic persons through direct contact; r i is the secondary infections generated by symptomatic persons through direct contact; r ae is the secondary infection seeded by asymptomatic persons through the environment; and r ie is the secondary infection seeded by symptomatic persons through the environment. we can also express r in terms of (t , c , q , where now, expressing the other state variables in terms of e, it implies that we now substitute a, i and v into second equation ( ) and factorizing e out gives from the initial hypothesis, e = and this implies that, making s * in equation ( ) the subject gives now, adding first equation and second equation of ( ), we substitute s * from equation ( ) and simplify e * we get where but, we know that the basic reproduction number is expressed as therefore, we express the endemic equilibrium points, (s * , e * , a * , i * , r * , v * ) in terms of r as; based on the preliminary notes above, we now state the lyapunov stability theorem. theorem (lyapunov stability theorem). the equilibrium, y * is globally stable if the function, l(y) is radially unbounded and positive definite globally such that it has globally negative time derivative, l(y) < ∀y = y * . we say that; the function l(y) is a lyapunov function if it satisfies the above theorem, the proof can be found in [ ] . another important theorem which also plays a key role here is the kransovkii-lasalle theorem. this is an extension of lyapunov function. in summary, this theorem puts forward that; considering an autonomous system, y = f (y) which has equilibrium, y * and that f (y * ) = , we assume there is a continuously differentiable positive definite and radially unbounded function l : r n → r which meets the conditionl(y) ≤ ∀ t, y ∈ r n . we then define the invariant set as proof. we employ the approach in [ ] to analyze both the stability at disease free and endemic equilibrium. we define a lyapunov, l for the disease-free equilibrium point as follows; differentiating l with respect to t gives; we substituteĖ,Ȧ,İ,v from equation ( ) intol gives; after further simplification we havė differentiating the function above gives; substituting equation ( ) into equation ( ) with further simplification gives; considering the expression we have that, h = . this implies that the coefficients of x x , x x and x x are all . equating the coefficients of x , x , x , x and x to and solving for h , h , h and h gives; therefore, t can be rewritten as it then follows that, t ≤ if x = , x = , x = , x = , x = and x = . hence we may conclude that;l by lasalle theorem, the invariant set is defined as since the invariant set, ζ only contains the endemic equilibrium (s * * , e * * , a * * , i * * , r * * , v * * ), then the endemic equilibrium is said to be globally asymptotically stable under the given region d. in this section, our focus is to verify the validity of the model. this is achieved by fitting and comparing the proposed model with a real data to know its degree of accuracy. are shown in table and figure respectively. the blue points in figure represent the cumulative number in computing for the normalized sensitivity index ( p r ) on the r for each of the parameters p, we use the formulae below [ ]; applying the formula above gives the parameters with their sensitivity index in the table . from table , asymptomatic class. the model shows that, asymptomatic individuals can join the severely infected class. however, the estimated parameter value shows that, only few people experience such situation, that is, . %. considering the system x (t) = f (x(t)) with x( ) = x , such that x ∈ r n where f : r n → r n and x : [ , ∞) → r n . we introduce variables responsible for the control u i , i = , , ...n ∈ n. we then have; the admissible control set is given as; u * = {u(t) ∈ l (t , t f )|u(t) ∈ a}. the aim here is to target the best control variables, u i , which can efficiently reduce the rate of secondary transmission at a minimum cost of their implementation at any time t ( ) ≤ t ≤ t (f ) [ ] . that is, we seek to achieve a reduction in the number of individuals in the susceptible, exposed, asymptomatic, severely infected classes and also reduce the content of the virus in the system at a minimum cost simultaneously. to achieve the above objective demands a lot of constructive considerations. for example, implementing total lock down for about two months as a control strategy in this context might highly prove not to be feasible. the loop hole here is; is the country adequately prepared both financially and technically to provide to the satisfaction of its inhabitants the basic needs such as food, water and others throughout the whole period assigned for this measure? it is highly probable the answer might be a big no. it is an undeniable fact that, this measure might prove impractical in controlling the spread in this country. this is why there is a need to objectively sort for more dense restrictive measures with flexible and feasible approaches to be employed in this setting so as to control the disease. we rely on the pontryagin's maximum principle as applied in [ ] for this analysis. we base on the premises above to set below likely control strategies: the objective functional under discussion, q, which is to be minimized is given as; subject to the constraints; from equation ( ) , we assume that, the weight constant of the exposed, infected (a and i classes) and the virus in the system is . also, to better observe and understand the influence of these control strategies on the model, we assumed that; no recovered individual is vulnerable to be reinfected in equation ( ). we accounted for the respective affiliated costs, b u , b u , b u , b u , b u , which are possible to be incurred during implementation where the square denotes their severity. it is very necessary to ensure that, the proposed optimal solution exists. for this reason, we employ filippove- cesari theorem as used in [ ] . in this case, we show that, the existence of the optimal control solution is . the convexity of the integrand of cost functional with respect to u on the set a [ ] . we now have the hessian matrix of the given cost functional as;  since the computed hessian matrix above is everywhere positive definite, it follows that, the objective functional, q(u , u , u , u , u ) is strictly convex [ ] . we also have that, given that the integrand of the objective functional, we now take into accounts the existence of the adjoint function λ i , i = , , ... , such that they satisfy the equations; with the transversality condition λ i (t f ) = given that ∀u i where i = , , ..., , we have the optimal control strategies with respect to the befitting variation argument is given as; we progress with the numerical simulations on the optimal control by using the estimated parameters in table . dynamics of the subpopulations of exposed, asymptomatic, and symptomatic individuals and the number of virus with control u and without any control implementation is demonstrated by figure . figure - figure shows the respective dynamics when control u , u and u is used separately. it is revealed here, cost-effectiveness analysis is carried out based on the numerical implementation of the optimality system conducted in section . the cost benefits associated with the implementation of the control strategies can be compared through cost-effectiveness analysis. thus, following the approach used in several previous studies [ , , , ], the incremental cost-effectiveness ratio (icer) is calculated to determine the most cost-effective strategy of all the different control intervention strategies considered in this work. most often, icer is employed to measure up the changes between the costs and the health benefits of any two different control intervention strategies i and j competing for the same limited resources. icer is defined mathematically as icer = difference in costs of control strategies i and j difference in infections averted by control strategies i and j . the numerator of icer in equation ( ) table . from table , it is observed that the value of icer( ) is greater than that of icer( ). this indicates that strategy is more costly and less effective than strategy . for this reason, strategy is excluded from the list of alternative control interventions competing for the same limited resources and icer is recalculated for strategies and as illustrated by table . it can be seen in table that icer( ) is greater than icer( ). this implies that the implementation of strategy is more costly and less effective than the implementation of strategy . hence, strategy is discarded from the list of alternative control intervention strategies competing for the same limited resources. now, the icer is finally recalculated for strategies and as shown in table . table reveals that icer( ) is greater than icer( ). hence, strategy is considered to be strongly dom- found to be globally asymptotically stable. we found that, the major transmission parameters β, β , m and m contributing to the basic reproduction number of . − . were all attributed to humans through personal contact with the susceptible class or activities with the environs. it is further inferred from this study that; applying optimal control strategy on the rate at which the virus is released into the system, m and m , and also on the relative transmission rate due to human behaviour will considerably strike down covid- pandemic. it was also found that, it might be possible the recovered individuals can be reinfected, see figure b . when this happens, then the number of the infected individuals will also increase. therefore, we highly recommend that, drug manufacturers should aim at drug samples which will induce permanent immunity in the recovered individuals so as to reduce the susceptible population. cost-effectiveness analysis was carried out based on the numerical implementation of the optimality system conducted in section . this showed that, cleaning of surfaces with home-based detergents is the most cost-effective strategy, followed by: the effective testing and quarantine when boarders are opened, the combination of all the controls then that of intensifying the usage of nose masks and face shields through education. it is highly guaranteed that, this study will help policy the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the parameter values (data) used to support the findings of this study have been described in section . disclosure the authors fully acknowledge that this paper was developed as a result of the first and second author's thesis and project work. novel coronavirus ( -ncov) situation report- novel coronavirus ( -ncov) situation report- novel coronavirus ( -ncov) situation report- novel coronavirus ( -ncov) situation report- novel coronavirus ( -ncov) situation report- novel 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of infectious and vector-borne diseases date: - - journal: j infect dis doi: . /infdis/jiaa sha: doc_id: cord_uid: udky ko the swiss development cooperation, canada’s international development research centre, the swiss tropical public health institute, and the unicef/united nations development programme (undp)/world bank/world health organization (who) special programme for research and training in tropical diseases (tdr) collaborated on a project to review, understand and promote the use of multisectoral approaches (msas) in the prevention and control of vector-borne diseases (vbds). the objectives of the project were to support a landscape analysis of how msas have been used in the prevention and control of vbds; to develop a theoretical framework for guiding the implementation of interventions; and to test the recommendations in real-life conditions. to realize these objectives, the project supported several activities, including commissioning a series of scientific reviews on msas in thematic areas, sharing the key findings of these reviews in workshops and events, and developing a guidance framework for the implementation of msas. these activities have produced the theoretical framework that will be tested in real-life conditions through the support of case studies. the collaboration on implementing multisectoral activities against vbds will continue among tdr, the swiss tropical public health institute, and new partners such as the who water sanitation and hygiene group, undp, and un-habitat, in order to face the challenges identified and propose solutions tailored to specific contexts. the prevention and control of vbds require strong and adapted msas with the full participation of all relevant sectors. the burden of infectious diseases has drastically decreased in the past years, but it still represents the major cause of premature death in the world and is expected to remain so until , with million deaths annually [ ] . vector-borne diseases (vbds), including malaria and emerging arboviral diseases, account for about one-quarter of all infectious diseases [ ] , and the significant progress against malaria are halting since a few years. further, the rapid expansion of other diseases. including those caused by arboviruses such as dengue, chikungunya, yellow fever, and zika, is shown by exponential increases in numbers of cases and fatalities. the emergence, transmission, and distribution of vbds are linked to a wide range of intertwined and partially overlapping factors that belong to multiple sectors-from the biological elements of the system, such as pathogen and vector characteristics, to social and global elements, such as poverty, human behavior, and climate change. it has become evident that the prevention and control of these diseases must be driven by more than a single approach, because transmission patterns are driven by vector-host-pathogen relationships in which natural conditions, human societies and vector parameters are dynamically interacting and changing. in this context, a multisectoral approach (msa) is required to effectively address these complex and dynamic transmission patterns. a first framework for msa against malaria was developed by the roll back malaria (rbm partnership) in collaboration with united nations development program (undp) through the multisectoral action framework for malaria (mafm), published in and revised in [ ] . the mafm calls for action at several levels and in multiple sectors, globally and across international and intranational boundaries, and by different organizations. it emphasizes complementarity, effectiveness, and sustainability, and it involves new interventions as well as putting new life into those that already exist, coordinating and managing these in new and innovative ways. with the purpose to expand the mafm to other vbds, the swiss development cooperation (sdc) and the swiss tropical public health institute (swiss tph) developed a concept note on which the current project was based. the sdc, the international development research centre (idrc) from canada, the swiss tph, and the unicef/united nations development programme (undp)/world bank/world health organization (who) special programme for research and training in tropical diseases (tdr) agreed on a collaborative activity, started in late , to better understand the landscape, the building blocks, and the processes of an msa for the prevention and control of vbds and to implement selected case studies to test these approaches. the objectives of this project were ( ) to synthesize global knowledge on current multisectoral activities that are deployed in different regions against infectious diseases, ( ) to draw theoretical processes and general recommendations from experiences, and ( ) to test the recommendations into real-life conditions through case studies. the project was then structured to include different steps ( figure ). the first step was the support of commissioned reviews on specific related topics; the second was the organization of joint meetings, workshops, and events to discuss and put together all relevant sectors; and the third was the development of a guidance framework for implementing of msas. these first steps have been fully completed in the past years and were used to draw the final step of the project. this last step is currently ongoing and will follow the implementation of case studies on msas against different diseases in low-and middle-incomes countries to test the theoretical framework. a call was launched in january to support commissioned reviews on specific topics related to msas for the prevention and control of vbds. the overall objective of the call was to support a landscape analysis on examples of msas to prevent and control vbd transmission and to identify knowledge gaps. the commissioned reviews were mandated to investigate current knowledge and experiences on topics related to different sectors. the publications on some of the main findings from the reviews are included in the current supplement to the journal of infectious diseases, and the abstracts from the final technical reports are provided below. review : impact of the industrial sector on vbds, with the example of gold mining activities disrupting malaria ecosystems in africa and the americas. the objective of this review was to retrieve and analyze available data and information on the impact of industrial activities on vbds transmission, with a special focus on mining activities that can greatly disturb existing malaria ecosystems in africa, asia and latin america. the information presented in this review has already been made available to improve policies, practices, and research priorities going forward [ ] . some key recommendations from the findings include establishing a national interministerial task force for vector control, promoting the building of suitable and location-specific multisectoral partnerships, mandating the use of health impact assessments and/or expanding the environmental impact assessments to health requirements, reaching the informal sector through local health posts and roaming healthcare workers, conducting risk mapping to determine transmission risks, ensuring community engagement and social outreach when implementing vector control programs, making health messaging and education a component of disease control efforts, and monitoring and evaluating vbd programs. including within the ecobiosocial context. weighted and prioritized integrated strategies for the prevention and control of vbds within the context of ecobiosocial approaches provided evidence for progressive implementation of more comprehensive ways to control vbds. high-level commitment of multiple ministries is central to the intersectoral interactions required to plan, fund, and implement prioritized activities outlined in the response. sustained political engagement will be required to maintain momentum for systems reforms required for adjustment to an integrated approach. prospective primary field studies are needed to generate evidence addressing the impact of integrated strategies to prevent and control vbds within the context of ecobiosocial approaches and the multisectoral participation. in addition, the usefulness of msa was found to go beyond current public health vbd threats to emerging and reemerging ones, especially viral diseases. population displacement and other forced movement patterns after natural disasters or armed conflicts or due to socioeconomic reasons were found to contribute significantly to the global emergence of aedes-borne viral disease epidemics. dengue and chikungunya epidemiology are critically affected by situations of displacement and forced movement patterns, within and across borders. in this respect, waves of human movements have been a major driver for the changing epidemiology and outbreaks of the disease on local, regional, and global scales. both emerging dengue autochthonous transmission and outbreaks in countries known to be nonendemic and cocirculation and hyperendemicity with multiple dengue virus serotypes have led to the emergence of severe disease forms, such as dengue hemorrhagic fever and dengue shock syndrome. the same was also found with atypical and severe forms of chikungunya when emerging in outbreaks due to human mobility and affecting nonimmune populations in new territories. the review was focusing on the vulnerable groups, such as the mobile and migrant population (mmp) in myanmar, which are now the focal malaria transmission groups, impeding malaria elimination in the country. to control malaria transmission and achieve subsequent malaria elimination, one of the interventions focused on increasing use of personal protective measures, such as insecticide-treated bed nets (itns) for the mmps in myanmar artemisinin resistance containment zones. the objectives of this study were to ( ) identify which stakeholders were involved in intersectoral approaches to support the intervention of increasing access to and use of itns targeted at the mmps in these zones, ( ) characterize the itn interventions targeted to these special groups of the population, and ( ) analyze how the intersectoral collaboration was deployed and how this approach was supported in the target population. the findings show that interventions to distribute itns for the prevention of malaria were supported by multiple stakeholders (local, national, nongovernmental organizations, and others); however, it was not described how the intersectoral collaboration was working. nevertheless, the net ownership and rates of use at the end of the project did not met the who targets for myanmar. further data were missing to specifically assess the role of the different stakeholders involved in the interventions. this review clearly demonstrates some gaps in looking at multisectoral collaboration, as well as the absence of specific indicators to show how msa was working and whether the failure was due to faulty implementation of the approach or to other factors. although who is recommending intersectoral collaboration as one of the key elements of integrated vector management and assumed this would make an important contribution to vbds control and elimination, there is limited evidence comparing the effect and contribution of intersectoral approaches with those of the health sector only. for that purpose, a systematic review from more than years of scientific literature was undertaken to develop an evidence-based framework of intersectoral collaboration and assess its effectiveness in sustaining the prevention and control of vbds. among the articles included in the analysis, were categorized as of moderately strong quality. all articles compared preintervention and postintervention outcomes against disease or vector variables (with the intervention being intersectoral collaboration). three articles included outcome variables on intersectoral collaboration and participation indicators. however, analysis by different sectors or different activities was retrieved. only article compared cost data for communityintersectoral intervention for indoors residual spraying (irs) and traditional "vertical" irs. six factors extracted from studies were identified as influencing the effectiveness of intersectoral collaboration. the main ones were using approach factor (in studies out of ), resources (in studies out of ), relationships (in studies out of ), and management (in studies out of of ). the recent global strategy of vbd control and prevention encourages intersectoral collaboration as an approach to achieving cost-effective and efficient results from an intervention. the review showed that although intersectoral collaboration has played an important role in achieving reduction of vbds or vector densities, very few studies have measured how much intersectoral collaboration contributed to this impact, and the relevant indicators are missing [ ] . review : scoping review of intersectoral collaborations to prevent and control vbds. this review synthesizes evidence for models of intersectoral collaborations for the prevention and control of vbds. half of the articles were about malaria control in the african region. the other half of the publications retrieved were on the prevention and control of dengue, with interventions based in asia and latin america. among the many gaps or challenges that impeded successful implementation and lowered the chances of project sustainability, most notably, the disconnect between different stakeholder responsibilities was often encountered and exemplified. the observation of a lack of communication between multilateral organizations and local governments, for whatever reason, is of great concern with a top-down approach that makes stakeholder relationships more susceptible to disconnection from field reality. the ownership of the programs by the community was an issue, as multiple cases cited the lack of understanding, interest, and initiative as a reason for discontinuity. this observation emphasizes the needs of msas, including local and community sectors. the lack of research capacity, including baseline data, skilled and knowledgeable staff, and models for data analysis that could be contextualized to local needs, was evident for both malaria and dengue control programs. the overall results show the need for a comprehensive framework for an effective and sustainable msa to prevent and control vbds. because both intersectoral collaboration and vbds are broad topics that hinge on social and economic development, the issues of financing, investment in human resource development, and supply of materials should be addressed through the collaboration. the delphi-validated statements summarizing recommendations for msas are globally applicable, but they need to be contextualized to a national and even municipal level. the workshop was attended by about participants and had a specific session with who member states. some activities were recognized by the participants as priority activities to be undertaken once the commissioned reviews have produced their results, through final technical reports and publications. these activities included specific work to support the msa for prevention and control of vbds and recommendations at different levels, including global, cross-border, national, and local. the recommendations from the workshop included the establishment of a guiding and advocacy framework as well as support for case studies to collect evidence on msa potential and functioning, including collection and sharing of data, analysis, cost estimates, overall health impacts, and risks for potential outbreaks. the recommendations for international agencies and national/local levels included a requirement for assessment of vector control needs; coordination of health impact assessment; and operationalization of the msa at the country level, creating a coordinated system that is ready to respond to different vbd situations, including when displaced people are affected or spread vbds. the aims of the session were an introduction to the rationale of msas for vbds; the presentation of key results from the commissioned reviews by the speakers; a discussion on successes, challenges, gaps, needs, and opportunities; and illustration of the potential beneficial impact of follow-up activities. the session began with a general introduction on the burden of vbds, the available evidence that msas are needed to prevent and control vbds needs msas, and the need to know where we are in conceptualizing and implementing these approaches. rashad abdul-ghani (phd) presented the review on chikungunya virus as a globally (re)emerging and rapidly expanding epidemic threat driven by human mobility patterns, showing that the expansion of this virus is strongly linked to human movement, which in turn is related to social, economic, political (multisectoral) factors. the warning that what is happening with chikungunya virus may also happen with any other virus, maybe more virulent, truly anticipated the current coronavirus disease pandemic. alfonso rodriguez morales (phd) presented the integrated strategies for the prevention and control of vbds within the context of ecobiosocial approaches, showing how to move from a single-oriented control of vbds (vector control for example) to a multisectoral one. the single-oriented control take into account only one sector and one approach such health sector and vector control, opposite to a multisectoral one taking into consideration more than one sector such health and water and more than a single approach such as vector control and water management. the added value of integrating traditional vector control activities (health system) with newer technologies (community involvement, social approaches) was discussed, along with how these activities and approaches can be synergized and the associated challenges. also discussed were recommendations on the way forward and potential opportunities for multisectoral research and action to help prevent and control vbds. jana fitria kartika sari presented intersectoral collaboration for the prevention and control of vbds, supporting the implementation of a global strategy, with some examples of success when working outside the health sector (eg, including education, agriculture, etc) and the remaining knowledge gaps. the final discussion introduced the global vector control response, which advocates for msas among the pillars of the response [ ] . diseases, " new orleans, louisiana, october . the annual meetings of the american society of tropical medicine & hygiene attract thousands of participants, so this was an ideal forum to expose this work to a wider audience before the planned peer-reviewed publication. the poster summarized the progress and suggested the expected outcomes and the next steps. this document [ ] was produced following one of the main recommendations from the reviews and exchanges with the stakeholders. the document starts with introductory chapters on vbd basics and msas. the determinants of vbds are described and grouped into the following categories: pathogen and vector related, environmental and agroecological, economic and social, and health system related. together, these determinants go beyond the ministries of health and the health sector, concerning many other sectors and stakeholders. existing prevention and control methods were laid out with challenges highlighted, among which was the weakness of the health sector alone. there are opportunities for better coordinated actions, such as potential synergy with the global momentum of the sustainable development goals, multiple entry points for interventions across sectors and diseases, and empowerment through more research. the historical background of msa was traced to reveal this inevitable growing consensus. examples of existing msa case studies were extracted from the commissioned reviews and briefly discussed. chapters and present the conceptual framework and its components. the conceptual framework is named bet-for base, energy, and technical elements-and includes components: pillars, dimensions, levels, resources, sectors, domains and enablers. these components envelope the ingredients to include in a customized and tailored msa. chapter outlines the coordination pathway from step to step . roles of nongovernment sectors and bodies are discussed with a focus on nongovernmental and international organizations, private sectors, and communities. because financing and legislation are an indispensable part of the entire coordination pathway, funding mechanisms and the types of norms and policies needed during a multisectoral collaboration are included in this chapter. the guidance also emphasizes integration and synergy with the existing institutional structure for msa within the country as well as with global multinational and multisectoral efforts, such as those under the health and environment linkages initiative, sustainable development goal-related programs, and the one health initiative. chapter covers sectoral guidance. a sectoral pathway is intended to assist government ministries to plan and initiate their work according to an msa, from defining the vision to sectoral monitoring and evaluation, including important steps such as aligning msa activities with the sector's existing activities. a nonexhaustive list of key sectors is included, along with health: environment, water and sanitation, agriculture and aquaculture, energy, housing, education and research, finance, and legislature. the concluding chapter of the guidance document highlights the need for a system to monitor and evaluate these approaches and the interventions. the activities developed around the analysis of msas for prevention and control of vbds have resulted in new evidences retrieved from the analyses of the available publications and findings on this topic. several thousands of articles were retrieved, screened, selected, and analyzed through the work done by the commissioned reviews. the findings were published and some of them are included in this special issue. the main results were discussed over several events and exchanges with stakeholders from different sectors and levels (from global to local). among the main challenges identified to building up an effective and efficient msa, the lack of pathway and framework was considered critical, but the development of such a theoretical framework was feasible and was then achieved. this guidance document has now been published and needs to be tested and improved in real-life conditions. although the guidance is primarily directed to decision makers, with specific relevance for governmental sectors, the framework can be tailored to suit the needs of subnational and decentralized stakeholders. the purpose of this framework is not only to support supraministerial leaders and health sector but also to enhance the capacity of decision makers in other sectors to achieve in a collective effort efficient prevention and control of vbds. because the guidance document aims to delineate "how to," apart from describing the essential components, recommendations are providedfor instance, on how to mobilize political will, how to enhance commitment and coordination among sectors, and how to strengthen community engagement. these elements will be studied in the following step of the project within specific cases of multisectoral collaboration to control specific diseases, such as malaria and arboviral diseases. for that purpose, research proposals will be supported through a partnership with one of the most relevant sectors linked to health and vbds, that is the water, sanitation, and hygiene (wash) sector. a proposal was developed in partnership with the who wash group to reduce wash-related disease of poverty with a primary focus on vbds, through the following activities: ( ) refining and promoting research for impact on multisectoral action for health, ( ) increasing the impact of joint convening of wash and health sectors, and ( ) supporting the strengthening of health systems to better address infectious diseases of poverty in general and vbds in particular. the project will include work packages: work package on strengthening the prevention and control of diseases of poverty through multisectoral collaboration, based on the latest research findings and who wash norms, and work package on strengthening health systems to better address infectious diseases of poverty through improved wash in healthcare facilities and enhanced capacity to manage wash services and engage in good hygiene practices. the partnership around the multisectoral activities will continue between tdr, the swiss tph, and new partners, such as undp and un habitat for building stronger recommendations and better addressing the numerous challenges identified. from past experiences and evidences, it has become clear that the prevention and control of vbds cannot be achieved and sustained through single-sector intervention(s) and without the full commitment of not only the responsible entities but also the communities involved. what msas are targeting is exactly this full participation of all relevant sectors according to their own incentives and needs. the international bank for reconstruction and development/ the world bank world health organization. global vector control response - . world health organization roll back malaria partnership to end malaria and united nations development programme. multisectoral action framework multisectoral approach to the prevention and control of vector-borne diseases: a conceptual framework the impact of industrial activities on vector-borne disease transmission intersectoral collaboration for the prevention and control of vector borne diseases to support the implementation of a global strategy: a systematic review key: cord- -ugeupo u authors: sim, shuzhen; ng, lee ching; lindsay, steve w.; wilson, anne l. title: a greener vision for vector control: the example of the singapore dengue control programme date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: ugeupo u vector-borne diseases are a major cause of morbidity and mortality worldwide. aedes-borne diseases, in particular, including dengue, chikungunya, yellow fever, and zika, are increasing at an alarming rate due to urbanisation, population movement, weak vector control programmes, and climate change. the world health organization calls for strengthening of vector control programmes in line with the global vector control response (gvcr) strategy, and many vector control programmes are transitioning to this new approach. the singapore dengue control programme, situated within the country’s larger vision of a clean, green, and sustainable environment for the health and well-being of its citizens, provides an excellent example of the gvcr approach in action. since establishing vector control operations in the s, the singapore dengue control programme succeeded in reducing the dengue force of infection -fold by the s and has maintained it at low levels ever since. key to this success is consideration of dengue as an environmental disease, with a strong focus on source reduction and other environmental management methods as the dominant vector control strategy. the programme collaborates closely with other government ministries, as well as town councils, communities, the private sector, and academic and research institutions. community engagement programmes encourage source reduction, and house-to-house inspections accompanied by a strong legislative framework with monetary penalties help to support compliance. strong vector and epidemiological surveillance means that routine control activities can be heightened to specifically target dengue clusters. despite its success, the programme continues to innovate to tackle challenges such as climate change, low herd immunity, and manpower constraints. initiatives include development of novel vector controls such as wolbachia-infected males and spatiotemporal models for dengue risk assessment. lessons learnt from the singapore programme can be applied to other settings, even those less well-resourced than singapore, for more effective vector control. vector-borne diseases (vbds) are a major cause of morbidity and mortality in the tropics and subtropics, accounting for more than % of the global burden of infectious diseases [ ] , with over % of the world's population at risk from at least one vbd [ ] . aedes-borne diseases, including dengue, chikungunya, yellow fever, and zika, are increasing at an alarming rate driven by urbanisation, local and global population movement, and climate change [ ] [ ] [ ] . aedes aegypti mosquitoes lay eggs in a wide variety of artificial containers and structures, including those that occur in discarded plastic containers, water storage containers, flowerpots, tyres, poorly constructed concrete structures in the ground, and gutters, which abound in urban environments. the main tool we have for controlling vbds is vector control [ ] . although vector control has been hugely successful against some vbds such as malaria [ ] , weak implementation of sustainable programmatic vector control has struggled to control aedes-borne diseases in many parts of the world in the past years. margaret chan, former director-general of the world health organization (who), famously said in her world health assembly address that the zika epidemic was "the price being paid for a massive policy failure that dropped the ball on mosquito control in the s" [ ] . in order to strengthen vector control, who member states called on who to develop the global vector control response - (gvcr) [ ] . this strategic document includes a framework of key priorities for vector control strengthening. the gvcr calls for collaboration within and outside the health sector, increased engagement of communities, scaling up and integration of vector control tools and approaches, and improved surveillance and monitoring and evaluation (fig ) . this is supported by a foundation of capacity and capability strengthening and by increased basic and applied research and innovation. also important are factors including country leadership, advocacy, resource mobilisation, and regulatory, policy, and normative support. who member states are reorienting their programmes in line with the gvcr [ ] , but good examples of the gvcr principles in action are currently limited. one exception is the dengue control programme in singapore, which in fact predates the gvcr but adopts many gvcr principles. this manuscript aims to document the key features of the programme, highlight how they sit within the gvcr framework, and draw out important lessons that can be applied by other vector control programmes, including those less well-resourced than singapore's. the republic of singapore is an island city-state in southeast asia with a land area of . km and a population of . million [ , ] . singapore is located at the southern tip of the malay peninsula, connected to peninsular malaysia via two causeways and by ferry services with indonesia's riau islands to the south. singapore is a travel and business hub with . million visitors in [ ] . the city-state is highly urbanised and has a high population density with almost , people per km , and typically tens of thousands of people per km in the main residential areas [ ] . since , singapore has been working towards the vision of creating a 'city in a garden', where greenery and biodiversity are seen as solutions to improving health and well-being of its citizens [ ] [ ] [ ] . seventy-nine percent of singapore's residents live in high-rise apartments built by the government's housing and development board (hdb), with the remainder living in privately owned condominiums ( %) or landed property ( %) [ ] . the climate in singapore is tropical and it is hot and humid all year round. singapore is endemic for dengue, with all four dengue virus (denv) serotypes circulating, frequent emergence of various genotypes, and a cyclical pattern of outbreaks every - years [ ] [ ] [ ] [ ] . in in , in , , and , singapore experienced explosive dengue fever outbreaks that resulted in , , , , , , and , indigenous cases, respectively, with incidence rates of . , . , . , and . per , population [ ] [ ] [ ] [ ] . in , singapore also experienced a large dengue outbreak with more than , cases [ ]. there is low herd immunity, particularly among younger generations, due to decades of low dengue transmission [ ] [ ] [ ] . the primary dengue vector is a. aegypti, and although aedes albopictus is also present, it is considered a weak secondary vector [ ] . exposure to chikungunya is low in singapore, with seroprevalence of . % among adults [ ] . the country has experienced small local outbreaks of chikungunya, with the largest ( , cases and an incidence rate of . per , ) occurring in , a year when aedes densities were high [ ] . singapore's first zika outbreak occurred in , and although it occurred at the same time as the epidemic in brazil and other latin american countries, the zika strain was found to have originated in southeast asia [ ] . malaria was eliminated in singapore in [ ] , but there are still sporadic imported cases and competent vectors including anopheles epiroticus (formally anopheles sundaicus), anopheles maculatus, and the emerging vector anopheles sinensis [ , ] . since independence, singapore has recognised that a clean and green environment is necessary not only to ensure good health and a good quality of life for its people but also for economic competitiveness. the government embarked on projects to clean up the land and waterways and invested heavily in critical sanitation and environmental infrastructure, including drainage development projects, sewerage and used water treatment infrastructure, and solid waste management. the environmental management put in place to implement this high standard of public cleanliness has greatly benefited singapore's efforts to tackle vbds. underscoring the view that aedes-borne diseases are environmental diseases, dengue control in singapore is led by the national environment agency (nea), a statutory board of the ministry of the environment and water resources (mewr). in view of the importance of infrastructure maintenance and design, environmental sanitation, people's behaviours, and use of technologies on dengue prevention, the nea collaborates closely with other government ministries (e.g., health, national development, education, finance), town councils (responsible for management and maintenance of the common property of public housing estates, including vector control), community associations, research and academic institutions, and the private sector (fig ) . intersectoral activities are coordinated by an inter-agency dengue taskforce which meets regularly, and monthly during outbreaks. in the event of a severe dengue outbreak, the taskforce membership is escalated to minister level. in addition to regular meetings, taskforce members are also in close contact via email and telephone to exchange feedback and information-for example, on unusual aquatic habitatsin a timely manner. resources for government agencies to carry out dengue control activities are allocated by the ministry of finance, to which each agency justifies its own funding needs. if necessary, nea supports these justifications, especially in an outbreak year when nea has called on agencies to step up dengue control measures. nea may also offer subsidies to town councils to support enhanced dengue control measures in public housing estates. for example, an important partner of the nea is the hdb, the statutory board responsible for public housing. in high-rise apartments in singapore, laundry is typically hung out to dry on bamboo poles fixed into tube-shaped bamboo pole holders on the side of the building. discovery of substantial numbers of aedes larvae in these holders led to provision of caps to cover them when not in use. later, the clothes-drying system was redesigned such that the bamboo poles now rest on brackets instead, eliminating the need for holders (fig ) . as mounting the bamboo poles in the holders required residents to lean out of the window, replacing the holders with brackets not only removed a mosquito breeding habitat but was also safer for residents. antimosquito valves that allow water to drain away but prevent escape of mosquitoes are installed in gully drains in hdb apartments, and hdb blocks are constructed without roof gutters, since these are difficult to access and maintain at height and therefore can become aedes habitats. nea works closely with town councils who are responsible for vector control around the hdb blocks. construction sites play an important role as a driver of sustained dengue transmission [ ] , since rainwater-filled land excavation holes, construction materials, and equipment (e.g., water tanks, skips, canvas sheeting) can become aedes habitats. the singapore contractors association limited (scal) is therefore an important partner for nea. construction sites are mandated to engage an environmental control officer (large sites may even have their own dedicated officer) who ensures appropriate action is taken to reduce vector proliferation, including larviciding with bacillus thuringiensis israelensis (bti) (fig ) . the land transport authority is responsible for good housekeeping and vector control on their infrastructure construction sites and the urban redevelopment authority conducts vector control along roads and their car parks. within the nea, the department of public cleanliness maintains a system of daily litter collection and its mandate is cleaning to 'public health standards'. approximately % of singapore's waste is recycled and the remainder incinerated, with the waste ash deposited on an offshore landfill site, semakau island, helping expand the island. this serves to reduce artificial containers in the environment which could otherwise become habitats for aedes mosquitoes. within the mewr, the nea works alongside the public utilities board (pub), which is responsible for ensuring a sustainable and efficient water supply and responsible for drainage system in singapore. vector control activities of the pub include designing drains for maintainability (accessible and with sufficient gradient to prevent water pooling) and regular flushing of drains monthly or bimonthly. the department of public cleanliness conducts regular cleaning of drains to remove any debris or refuse. the national parks board (nparks) also works closely with nea to maintain a pleasant environment with trees, plants, and water features such as ponds and streams, while keeping vector densities to a minimum. vector control measures implemented include weekly bti treatment of ponds and regular removal of floating vegetation. if vectors do become a problem, surveillance and vector control measures are quickly put in place. for example, in the first quarter of , ornamental papyrus beds in bishan-ang mo kio park encouraged the proliferation of biting midges and a. sinensis. nea's identification of the vectors responsible led to control measures including bti application, intermittent drying of the papyrus beds, and the nea corporate communications department is responsible for mass and social media communications, whereas the p (people, private, public) network division leads nea's education and publicity initiatives and programmes for the p sectors, across all environmental initiatives, not just dengue prevention. community engagement makes use of existing structures including the people's association (the statutory board responsible for promoting social cohesion) and grassroots organisations under the people's association, such as citizens' consultative committees, residents' committees in hdb estates, and neighbourhood committees in private housing estates. these groups encourage bottom-up participation by seeking residents' cooperation in checking for mosquito breeding in homes. nea also trains members of the public and the people's association's community emergency response teams as dengue prevention volunteers, who educate fellow residents on dengue prevention. each year the p network division organises the national dengue prevention campaign, with the timing of the launch dependent on the dengue forecast provided by the environmental health institute (ehi), the research arm of nea. p work through the mayors of each district, grassroots members, and dengue prevention volunteers who mobilise their communities to conduct source reduction according to the ' -step mozzie wipeout' by doing house-to-house visits, distributing educational materials, and organising block parties and other events. the ' -step mozzie wipeout' targets the top five most common habitats in residential premises in singapore. information materials are available in all four written national languages (fig ) . the ' -step mozzie wipeout' recommends to ( ) turn the pail, ( ) tip the vase, ( ) flip the flowerpot plate, ( ) loosen the hardened soil, and ( ) clear the roof gutter and place bti larvicide inside. for those interested in aedes control outside singapore, it is important to note that these aquatic habitats may not be the top five habitats in other countries, so it is important to survey local sites to identify the major sources of a. aegypti. in singapore, messaging is directed towards people taking personal responsibility for mosquito prevention and explaining that by conducting source reduction, they can protect their families from dengue. p uses different methods to evaluate knowledge, attitudes, and practices of communities over time and looks for fresh angles to prevent fatigue from regular public health messaging. besides nationwide general messaging, community engagement strategies also target specific population groups to encourage them to play a greater role in mosquito prevention, including domestic helpers, construction workers, the elderly, and school children. for example, domestic helpers and construction workers are targeted with behaviour change messaging through outreach and roadshows at dormitories, shopping malls, and other places of congregation. since these groups are often migrants and therefore transient populations, behaviour change materials are produced in the relevant languages (e.g., bahasa, hindi, indonesian, tagalog) and outreach is conducted regularly. dengue prevention roadshows are also conducted at elderly care corners and videos are available in local chinese dialects used by many elderly residents who originate from mainland china. the p network division partners with the ministry of education to include dengue prevention in school curricula at primary, secondary, and tertiary levels. timely information on the number and location of dengue cases is made publicly available on the nea website and myenv mobile app. nea also implements a community dengue alert system, in the form of colour-coded banners placed in high-visibility locations in dengue clusters to inform residents about the dengue situation in their neighbourhood and the corresponding actions they can take (fig ) . the banners use the widely recognisable 'traffic light' colour code of red (high alert), yellow (medium alert), and green (low alert) to allow for easy interpretation of the situation. communities are engaged and regularly give feedback about, for example, increased mosquito numbers, littering, and other environmental issues either online or via a -hour nea hotline. globally, aedes indices such as the house index (hi, percentage of houses positive for larvae and or pupae) are traditionally used for aedes vector monitoring, with a value of % often being used as an arbitrary dengue transmission threshold [ , ] . the singapore dengue control programme has brought the hi to very low levels (from % in the s to . % in the s). despite this low hi, singapore still experiences regular outbreaks, suggesting that the hi is no longer sensitive for dengue risk assessment [ , ] . with low vector populations, the singapore programme now uses in-house-developed gravitraps, which are designed to lure and trap gravid female aedes on the sticky lining [ ] . since , a system of , gravitraps have been deployed in public housing estates nationwide, and the scheme will be expanded to landed housing by end of [ ] . monitoring of the traps every weeks is currently outsourced, although an automated trap is currently under development by nea. gravitrap indices are plotted on a geographical information system (gis) and used to target intensified source-reduction campaigns. areas with a high gravitrap index are also published on the nea website to motivate the local community to take action to reduce dengue transmission. in keeping with the country's green aspirations, insecticides are used judiciously, with bti and temephos used as larvicides and pirimiphos-methyl as adulticide. choice of insecticide is guided by resistance monitoring, which is performed every few years. as well as aedes, surveillance is also conducted for culicines (potential japanese encephalitis vectors) and anophelines using the in-house-developed night catcher, a modified cdc light trap that keeps caught adult mosquitoes fresh and alive for analysis and segregates hourly-caught mosquitoes in separate containers. the ministry of health is responsible for case surveillance and clinical management of dengue patients. dengue is a notifiable disease, meaning that medical practitioners and clinical laboratories must report clinically suspected and laboratory-confirmed cases. if a patient presents at a health facility with suspected dengue, then blood samples are taken for diagnostic testing at a hospital or private laboratories using either rapid diagnostic tests against nonstructural protein (ns ) and immunoglobulin m (igm) antibodies or reverse transcriptase polymerase chain reaction (rt-pcr) for acute cases. alternatively, samples can be sent to the ehi for diagnosis, and through this system, ehi is able to monitor circulating dengue serotypes [ ] . an epidemiological investigation of each case, conducted by telephone or in person, is triggered by the environmental public health operations department at nea, and case locations are plotted on a gis interface. a dengue cluster is formed when two or more cases have onset within days and are located within m of each other (based on residential and workplace addresses, as well as movement history collecting during the epidemiological investigation). dengue clusters are graded and the situation communicated to the public via the colour-coded community dengue alert system: high-risk area or more cases (red); high-risk area with fewer than cases (yellow); and no new cases, under surveillance for the next days (green) (fig ) . once a cluster is formed, this triggers the nea environmental public health operations team to ramp up vector control activities in the cluster. entomological indices and dengue case numbers are not reliable measures for assessing the long-term impact of vector control programmes because of changes in surveillance and diagnostic capabilities over time [ ] . instead, nea uses blood bank igg seroprevalence to estimate the force of infection (foi) over time [ ] . the singapore dengue programme relies predominantly on source reduction and larviciding using bti, which are implemented throughout the year through community and programme efforts. in the first months of , approximately % of vector habitats were found in residential premises, rising to % in cluster areas. house inspections are conducted routinely and at increased frequency in cluster areas by the environmental public health operations team (fig ) . field staff follow a strict protocol including identifying themselves clearly and ensuring that the resident witnesses the taking of any samples for species identification at the ehi. use of insecticides is seen as a short-term solution, so fogging with organophosphates is restricted to clusters during outbreaks only. source-reduction activities are facilitated by legislation and law enforcement, which singapore uses in addition to community engagement to enhance public compliance. according to the control of vectors and pesticides act (cvpa, the main legislation dealing with mosquito breeding), the operations team can enter homes to conduct inspections and vector control, and residents are fined at least s$ (us$ ) if aquatic stages of vectors are found on their premises [ ] . inspections of construction sites and other premises are also conducted by a dedicated nea team. if aquatic stages of the vector are found, then an order can be served for vector control to be implemented within a specific time period by a registered vector control personnel. in the event that environmental management is found wanting, the cvpa allows nea to issue an order to stop any work being undertaken on the premises indefinitely or until vector control to remove favourable habitats has been carried out. a list of construction sites that currently have stop work orders are available on the nea website (www.nea.gov.sg). failure to comply with the order can lead to a fine of up to s$ , (us$ , ) and, in the case of a second or subsequent conviction, to a fine of up to s$ , (us$ , ) and/or imprisonment for up to months. the cvpa also covers pesticides registration, as well as licensing and certification of private-sector vector control operators [ ] . to address challenges such as climate change, increasing urbanisation, and the manpowerintensive nature of source-reduction activities, the singapore programme invests in research and development of new vector control technologies. for example, ehi is evaluating the use of wolbachia, a maternally inherited endosymbiotic intracellular bacterium, which when artificially introduced into a. aegypti can suppress dengue infections. singapore opted for a population-suppression strategy whereby only male wolbachia mosquitoes are released. when male wolbachia mosquitoes mate with wild-type females, the eggs produced will not hatch, because of a phenomenon called cytoplasmic incompatibility. use of suppression was favoured over a population-replacement strategy, in which both males and females are released, since it is in line with the long-term programme strategy of source reduction. wolbachia testing has followed a phased approach in two main release sites, including gaining an understanding of the basic biology of the released male wolbachia mosquitoes in the field, such as how far and how high they fly. the programme has also collaborated with the international atomic energy agency (iaea) to irradiate wolbachia mosquitoes at the pupal stage to render infertile any remaining females, so as to avoid replacement of the wild-type population with wolbachia a. aegypti mosquitoes. pilot testing has been accompanied by a careful community engagement campaign developed by ehi to emphasise that wolbachia is safe and that male mosquitoes do not bite, and to encourage continued source reduction. the programme also works with private-sector collaborators to develop and evaluate technologies for wolbachia testing and implementation. ehi works with orinno technology, a local start-up company, to develop new automated equipment to facilitate their work, including a larvae counting system, pupae sorting and counting system, and mosquito launcher to simplify and speed up the releases of the wolbachia mosquitoes by field staff. the programme also works with verily life sciences, an alphabet, inc., affiliate, to field-test the company's automated sorting and release technologies. the dengue control programme has developed a novel indicator for entomological risk assessment known as the a. aegypti breeding percentage [ ] . routine larval surveillance is not uniform spatially and temporally and so would be biased if used for risk assessment. instead, the breeding percentage expresses the number of a. aegypti-positive habitats over the total number of aedes-positive habitats (a. aegypti and a. albopictus) to cancel out the sampling error from nonsystematic inspection and cryptic breeding sites. predicting dengue outbreaks is difficult and the ehi has, in partnership with academic and research institutions including the national university of singapore, developed several different risk models to enable better resource planning and preparedness for outbreaks. for example, a risk map is prepared each year to guide resource allocation to different areas [ ] , a temporal model is used to predict dengue cases up to months in advance [ ] , and a spatiotemporal model integrating climate, vector density, population demographics (connectivity using public transport and mobile phone data), cases, infrastructure (age of building and number of units), and satellite data (vegetation) is used for high-resolution prediction and realtime allocation of resources [ ] . staff personal and professional development is a focus of the nea. for example, staff undertake continuing professional development courses organised by the internal training arm of nea, the singapore environment institute. rotations between departments are encouraged and staff can receive financial support and leave of absence to attend further education. there are possibilities for both vertical and horizontal movement within the organisation, recognising the need for both specialists and generalists. the ehi partners with academic and research institutions, and staff members have obtained phd and other degrees through their research work conducted at the ehi. the ehi of nea has been a who collaborating centre for reference and research of arbovirus and their associated vectors since . this involves consulting and advising (e.g., the director of ehi sits on the who strategic technical advisory group on neglected tropical diseases and strategic advisory group of experts working group for dengvaxia, the sanofi dengue vaccine), enhancing global outbreak preparedness (e.g., cross-border virus surveillance through unitedengue consortium, evaluation of diagnostics, etc.) and capacity building (e.g., training and sharing of best practice). the singapore dengue control programme is one of the best in the world, but what makes it so successful, and how can the lessons learnt be applied to other vector control programmes? although in many countries dengue control sits under the ministry of health, unusually in singapore it sits within the mewr. this is in line with the programme view that dengue is an environmental disease. dengue vector control uses mainly environmental management approaches, such as proactive source reduction, and environmental management including drainage and house improvements contributed to the elimination of malaria from singapore in [ ] . key individuals including the director-general of public health, could be trained as engineers or other professionals, not medical doctors. a strong sense of environmentalism stems from singapore's founding father, lee kuan yew, who oversaw the transformation of singapore after independence and in championed the idea of the 'garden city' [ ] . lee kuan yew promoted a green environment that was free of litter in order to create good living conditions for singapore's residents, but also to simultaneously encourage tourism, investment, and trade. strong political will and long-term political stability (the people's action party have been the only party to form a government since independence in ) means that this vision and trajectory has been maintained over time, for example, in the current sustainable singapore blueprint [ ] . despite sitting within mewr, the programme collaborates closely with the ministry of health with efficient systems for sharing information on confirmed cases to allow rapid intervention by the nea environmental public health operations team. adopting a similar environmental approach to vector control could enable more effective control of vbds worldwide (and incidentally was largely responsible for the success of vector control in the early and mid- s before the advent of ddt [ ] ). for example, progress in controlling malaria is stalling in many high-burden countries due to weak vector control programmes, and potentially also insecticide resistance [ , ] . since malaria is primarily a disease caused by standing water, proactively tackling immature vectors by using environmental management could be a synergistic addition to predominantly insecticide-based adult anopheline control. increasingly complicated and multidimensional public issues including climate change, globalisation, public health, and infectious disease outbreaks call for a transformation in public administration. since the s, singapore has adopted a whole-of-government approach, which 'denotes public service agencies working across portfolio boundaries to achieve a shared goal and an integrated response to particular issues' [ ] [ ] [ ] . the whole-of-government culture, propagated for decades in singapore, facilitates the view of dengue control as a shared responsibility across agencies. for example, cross-sectoral collaboration for dengue control is facilitated by the inter-agency dengue taskforce. further, rotation of leadership between different government departments and agencies means that so-called 't-shaped' managers have not only specialist expertise but a broader perspective on issues and can help to break down departmental or agency silos [ ] . this coordinated whole-of-government approach is exemplified by the singapore response to a dengue outbreak that coincided with the start of the covid- pandemic in early (box ). singapore also employs collaborative governance, whereby governing is based on collaboration between government and nongovernment stakeholders. this is exemplified by the important role of the people's association in bridging between government and communities. community outreach initiatives enjoy broad political support, as they provide an opportunity for the government to directly connect with the community. local politicians are also invested in preventing outbreaks in their constituencies and often communicate the importance of dengue prevention to residents during walkabouts. another success factor may be the use of a 'carrot and stick' approach to source reduction with community engagement and behaviour change campaigns led by the p network division, backed up by strong legislation and enforcement. a recent study conducted by ehi shows that houses that have more frequent inspections have a lower number of reported mosquito larval habitats [ ] , lending support to the system of house inspections. a lack of corruption (singapore is rated as one of the least corrupt nations in the world by transparency international [ ] ) also supports the penalty system for vector habitats. the clear accountability of mosquito breeding offences under the law makes it easier for stakeholders to understand their respective roles, thus facilitating collaborative action against mosquito breeding. strict enforcement of the law is another push factor that encourages joint efforts to take preventive measures. imposition of fines for vector habitats may not be possible in all vector control programmes globally but should be considered. singapore's dengue control response in , which is taking place amid the covid- pandemic, offers a case study of intra-and intersectoral collaboration to combat twin environmental public health threats. detection of dengue threat and raising the alert more than , dengue cases were reported in the first quarter of , double that for the same time period in [ ] . in early , ehi's risk models, incorporating case data from the ministry of health, forecasted a dengue surge in the coming months. ehi's analysis of serotype trends further detected an increase in the proportion of denv- cases, with denv- overtaking denv- as the predominant serotype in early . as denv- has not been predominant in singapore for nearly three decades, population immunity to this serotype is likely low, further increasing the risk of an outbreak. given this outlook for , nea alerted relevant government agencies and set in motion an enhanced and coordinated dengue control response. with support from political and grassroots leaders, nea brought forward the national dengue prevention campaign (typically held just before the traditional midyear peak dengue season) to late march [ ] , with the intention of raising awareness and rallying the public to conduct preemptive measures early on. this kicked off island-wide community-led outreach efforts, helmed by grassroots leaders and supported by dengue prevention volunteers, to encourage residents to carry out dengue prevention practices. at the organisational level, the nea-led inter-agency dengue taskforce met in january and march to coordinate the response across sectors and continues to meet regularly. emphasis has been placed on enhancing vector control in assets managed by various agencies (such as buildings, reservoirs, drains, and parks), especially if these are located in areas with high mosquito populations or within dengue clusters. singapore reported its first case of covid- on january [ ] . to curb local covid- spread, nea in february launched "sg clean" [ ] , a whole-of-government campaign to rally individuals and businesses to keep public areas clean (such areas include toilets, hawker centres, community spaces, and other premises). dengue control messaging was woven into 'sg clean'. a key campaign message was that enhanced public cleanliness, such as maintaining clean premises and not littering, eliminates mosquito breeding habitats and hence helps to reduce the spread of dengue in addition to covid- . in april , singapore implemented a 'circuit breaker' to curb covid- transmission, which involved stringent social distancing measures and cessation of nonessential work activities [ ] . given the high-risk dengue outlook for , nea worked at the whole-of-government level to include vector control activities as an essential service to what continues to drive transmission in singapore, where there is a well-resourced and effective dengue control programme? the dengue incidence rate in singapore has increased dramatically in the last years, but this is likely because of improved diagnostics, increased referral for testing by medical practitioners, and increased awareness among the public [ ] . a better indicator of the true infection rate is dengue foi: between the s (when singapore first implemented environmental management and vector control programmes) and the s, the dengue foi in singapore dropped -fold to approximately . ( per , individuals per year) and has since held steady at this low level [ ] . the decline in foi can probably be attributed to the effectiveness of the dengue control programme (along with an increasingly ageing population). this success in reducing disease transmission has resulted in a lowered herd immunity against dengue, leaving singapore's population vulnerable to outbreaks despite a low vector population. another challenge is the high level of population movement in and out of the country, which is known to facilitate the co-circulation of denv serotypes [ ] . singapore has , km of expressways and . km of mass rapid transit (mrt) lines across the island; over , people commute into the island state from malaysia each day [ ] ; and in , there were . million passenger movements in and out of changi airport from cities in countries and territories worldwide [ , ] . in order to reduce the foi further, the programme is evaluating innovative strategies such as wolbachia, novel community engagement mechanisms, and risk mapping for more effective intervention targeting. as well as better implementation of current tools and approaches, new vector control tools are urgently needed to combat vbds worldwide. in conclusion, the dengue control programme in singapore provides an excellent example of the gvcr in action. important elements include strong collaboration across government departments and between government and nongovernment actors, integration of tools and approaches, effective surveillance, and community engagement. as a high-income country, singapore is in an enviable position of having reliable government funding for dengue control. nevertheless, aspects such as working across sectors or implementation of environmental management do not need to be expensive, and the former can even save costs for the vector control programme. adoption of these elements could lead to more effective vector control programmes worldwide to reduce the intolerable burden of vbds. be continued during the 'circuit breaker' period. businesses and owners of premises are expected to ensure that adequate vector control activities continue at their premises (including offices, commercial buildings, schools, and construction sites), even if regular operations are on hold. nea continues to carry out island-wide routine inspections and enforcement, with precautions taken to minimise covid- transmission. these precautions include ensuring that officers carrying out inspections are healthy, wear masks, and practise good personal and hand hygiene. despite the efforts of the nea, dengue control has been particularly challenging in because of the switch to denv- , warm weather, and high numbers of people staying at home during the 'circuit breaker' period, which can increase aedes aquatic habitats and provides easy access to blood meals for female aedes mosquitoes. we thank the following individuals for their insights: dulcie chan, rama chandramogan, khoo seow poh, christina liew, sueanne mocktar, ong chin soon, tai ji choong, tony teo, and grace yap of singapore's national environment agency and nanthini elamgovan of singapore's national parks board. thanks also to manuela bernardi for her assistance in producing the figures. • vbds are environmental diseases-situation of programmes within the ministry of environment and/or strengthening environmental management is encouraged. • whole-of-government approaches (working across government ministries) and collaborative governance (collaboration between government and nongovernment) support vbd control. • strong vector and epidemiological surveillance enables targeting of vector control interventions. • consider the role of legislation and enforcement in reducing vector habitats. • stable financing supports effective vector control. • innovation and science-based approaches should be harnessed to support surveillance and control. top five papers world health organization. vector-borne diseases fact sheet integrating vector control across diseases the global burden of dengue: an analysis from the global burden of disease study urbanization and globalization: the unholy trinity of the st century the current and future global distribution and population at risk of dengue the importance of vector control for the control and elimination of vector-borne diseases the effect of malaria control on plasmodium falciparum in africa between address to the sixty-ninth world health assembly by director-general of the world health organization world health organization. global vector control response world health organization. framework for a national vector control needs assessment geneva: who online database-total land area of singapore world bank. population density (people per sq. km of land area) ministry of national development-centre for liveable cities singapore. sustainable singapore blueprint forging a greener tomorrow: singapore's environmental journey from slum to eco-city singapore's journey towards environmental and water sustainability singapore: department of statistics singapore forecast of dengue incidence using temperature and rainfall epidemiological aspects of an outbreak of dengue fever/dengue haemorrhagic fever in singapore the dengue haemorrhagic fever outbreak in singapore and its control dengue in singapore from to : cyclical epidemic patterns dominated by serotypes and communicable diseases surveillance in singapore communicable diseases surveillance in singapore communicable diseases surveillance in singapore communicable diseases surveillance in singapore environment agency. quarterly dengue surveillance data-q , q , q and q dengue seroprevalence of healthy adults in singapore: serosurvey among blood donors seroepidemiology of dengue in the adult population of singapore force-of-infection and true infection rate of dengue in singapore-its implication on dengue control and management gravitraps for management of dengue clusters in singapore seroprevalence of antibodies against chikungunya virus in singapore resident adult population hard lessons in surveillance and response from the singapore zika experience eradication of malaria from singapore risk of anopheles sinensis as an emerging malaria vector in singapore a study on anopheles maculatus and anopheles sundaicus in singapore construction sites as an important driver of dengue transmission: implications for disease control critical examination of aedes aegypti indices: correlations with abundance assessing the relationship between vector indices and dengue transmission: a systematic review of the evidence dengue prevention and years of vector control in singapore who regional office for the western pacific. guidelines for dengue surveillance and mosquito control. manila: who regional office for the western pacific dengue in singapore from to : cyclical epidemic patterns dominated by serotypes and control of vectors and pesticides act a novel entomological index, aedes aegypti breeding percentage, reveals the geographical spread of the dengue vector in singapore and serves as a spatial risk indicator for dengue. parasit vectors mapping dengue risk in singapore using random forest three-month realtime dengue forecast models: an early warning system for outbreak alerts and policy decision support in singapore memoirs of lee kuan yew. singapore: times media private limited connecting government: whole of government responses to australia's priority challenges. canberra: management advisory committee (australian government) a primer on implementing whole of government approaches. dublin: centre for effective services reviewing whole-of-government collaboration in the singapore public service introducing t-shaped managers. knowledge management's next generation nea brings forward national dengue prevention campaign and rolls out additional new tools to combat dengue, with increasing evidence of a sustained switch in dengue virus serotype confirmed imported case of novel coronavirus infection in singapore: multi-ministry taskforce ramps up precautionary measures campaign launched to rally public and businesses to work together to keep singapore clean circuit breaker to minimise further spread of covid- the effectiveness of inspections on reported mosquito larval habitats in households: a case-control study transparency international secretariat increasing airline travel may facilitate cocirculation of multiple dengue virus serotypes in asia dengue outbreaks in singapore and malaysia caused by different viral strains key: cord- -lnv yi l authors: perkins, alex; espana, guido title: optimal control of the covid- pandemic with non-pharmaceutical interventions date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: lnv yi l the covid- pandemic has forced societies across the world to resort to social distancing to slow the spread of the sars-cov- virus. due to the economic impacts of social distancing, there is growing desire to relax these measures. to characterize a range of possible strategies for control and to understand their consequences, we performed an optimal control analysis of a mathematical model of sars-cov- transmission. given that the pandemic is already underway and controls have already been initiated, we calibrated our model to data from the us and focused our analysis on optimal controls from may through december . we found that a major factor that differentiates strategies that prioritize lives saved versus reduced time under control is how quickly control is relaxed once social distancing restrictions expire in may . strategies that maintain control at a high level until summer allow for tapering of control thereafter and minimal deaths, whereas strategies that relax control in the short term lead to fewer options for control later and a higher likelihood of exceeding hospital capacity. our results also highlight that the potential scope for controlling covid- until a vaccine is available depends on epidemiological parameters about which there is still considerable uncertainty, including the basic reproduction number and the effectiveness of social distancing. in light of those uncertainties, our results do not constitute a quantitative forecast and instead provide a qualitative portrayal of possible outcomes from alternative approaches to control. the first known case of covid- in the united states arrived from china on january , [ ] . in the weeks that followed, deficiencies in testing allowed the virus that causes this disease to spread largely undetected [ ] , with the number of reported cases growing to , by april , [ ] . social distancing-such as closing schools, working from home, and sheltering in place-has been adopted widely and appears capable of impacting transmission [ , ] . these practices have tremendous economic and social consequences, however, which has precipitated growing desire to relax them [ ] . a danger in relaxing the use of social distancing is that the virus could resurge in its absence [ ] . a safe, effective, and widely used vaccine would obviate the need for social distancing, but there are many challenges associated with developing a vaccine against covid- [ ] and one is not likely to be available until at least spring [ ] . strategies for successfully controlling covid- until then will depend on a suite of non-pharmaceutical interventions (npis) [ ] , including some degree of social distancing but also diagnostic testing, contact tracing, and case isolation [ ] . in models of pathogen transmission based on mass-action assumptions, npis all act in a similar way by reducing the transmission coefficient. this parameter, often denoted β, reflects the product of the rate of contact between susceptible and infectious people and the probability of transmission upon contact [ ] . due to the extent to which they reduce contact, full-scale lockdowns appear to be the most effective strategy for reducing transmission available at present [ ] . until alternative npi-based strategies can be implemented that are similarly effective but less disruptive economically, it is urgently important to determine the minimal extent to which contact must be reduced to achieve public health objectives. optimal control theory offers a way to understand how to apply one or more time-varying control measures to a nonlinear, dynamical system in such a way that a given objective is optimized [ ] . these techniques have been widely applied to a variety of pathogen transmission systems before (e.g., [ , , , ] ), including in the context of a pandemic of a respiratory pathogen (e.g., [ , , ] ). the latter studies indicate that the level of npi-based control required is dependent on model parameters, and that application of npis can be required at a high level and for a long duration in the absence of a vaccine. here, we apply optimal control theory to determine optimal strategies for the implementation of npis to control covid- , with a focus on the us. an optimal strategy in this sense involves weighing the relative costs of control and covid-related mortality, and finding an approach to control that minimizes that combined cost. other optimal control analyses of covid- are beginning to emerge [ , , , , ] , although these have been less focused on any particular geographic setting. one contextual feature of the us that our analysis considers is the level of control that was implemented early in the pandemic. we account for this, because our objective is less about understanding what level of control would have been optimal earlier on and more about understanding what level of control would be optimal going forward, conditional on what has happened already. in addition, we emphasize the sensitivity of our results to epidemiological parameters that are not yet well characterized but appear influential in determining what range of impacts of npis on covid- are possible. all code and data used in our analysis is available at http://github.com/talexperkins/covid optimalcontrol. we modeled sars-cov- transmission according to a system of ordinary differential equations, with variables and parameters defined in tables and . in the model, all individuals are initially susceptible, s, with infections introduced at a constant rate, ι, from outside the population. individuals then transition to the exposed class, e, where they reside for an average of ρ − days. a proportion σ experience a symptomatic infection, i. the remainder experience an asymptomatic infection, a, and have a fraction, α, of the infectiousness of symptomatic infections. individuals reside in the i and a classes for an average of γ − days. all asymptomatic infections and a proportion relationship between the number of hospitalizations and the probability of death from covid- among hospitalized patients. the parameters ∆ − and ∆ + represent lower and upper bounds on the probability of death, and hmax represents the hospital capacity above which the probability of death exceeds ∆ − . hospitalizations are quantified as a proportion of the overall population. − κ of symptomatic infections then recover and become fully immune to subsequent infection. the remaining proportion κ of symptomatic infections transition to the hospitalized class, h, from which they exit through either recovery or death after an average of η − days. rather than track deaths due to covid- , d(t), as a state variable in eqn. ( ), we assume that they follow directly from hospitalizations, h(t), according to this results in the probability of death moving beyond a minimum of ∆ − towards a maximum of ∆ + as h(t) exceeds h max , as illustrated in figure . the motivation for this choice is to account for the possibility that patients could experience increased mortality when the demand for certain resources, such as intensive care unit beds or ventilators, exceeds their availability. one other optimal control analysis of covid- has incorporated a similar phenomenon [ ] , albeit with a different functional form. individuals who are recovered and immune are not followed explicitly, as the model's assumption of density-dependent transmission only requires spec-. cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint ification of susceptible and infectious classes in the transmission term. due to our assumption that rates of birth, µ, and death due to reasons other than covid- , δ, are equal, changes in population size over the course of the epidemic are modest, making the distinction between density-and frequencydependent transmission negligible. the primary form of control in the model is achieved through the variable u, which represents a proportional reduction in the transmission coefficient, β. as is standard for mass-action models of directly-transmitted pathogens, β reflects the product of the rate at which susceptible and infectious individuals come into contact and the probability of transmission given that a contact has occurred [ ] . thus, a wide range of npis could result in changes in u, including school closures, work from home policies, and shelter in place mandates, as well as more targeted approaches, such as isolation based on self-awareness of symptoms or contact tracing. in addition to control through u, the v class represents individuals who have been vaccinated, with individuals entering v from s at rate ν beginning on day τ ν . we assume that vaccination may not provide complete protection, resulting in vaccinated individuals becoming infected at a fraction − of the rate at which fully susceptible individuals become infected. at its core, the behavior of this model is similar to that of an seir model with demography. because analyses of the transient and asymptotic properties of this class of models are plentiful in textbooks and elsewhere (e.g., [ ] ), we omit such an analysis here. we do, however, derive a formula to express the basic reproduction number, r , as a function of model parameters. this relationship plays a role in how we parameterize the model. we use the next-generation method [ ] to obtain a formula describing r as a function of model parameters. this method depends on matrices f and v, whose elements are defined as the rates at which secondary infections increase the i th compartment and the rates at which disease progression, death, and recovery decrease the i th compartment, respectively. for "disease compartments" e, a, i, and h, these matrices are defined as these matrices are then used to define two others, . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . where x represents disease compartments and y non-disease compartments. the inverse of v is which, along with f , defines the matrix k = f v − . specifying we obtain as the maximum eigenvalue of k. the optimal control problem is to minimize the objective functional subject to the constraints of the state dynamics described in eqn. ( ) and the initial conditions that s(t ) = and all other state variables equal zero at time t . the parameter c weights the extent to which the control, u(t), is prioritized for minimization relative to deaths, d(t). squared terms for d(t) and u(t) are chosen both for mathematical convenience in the case of u(t) and to more heavily penalize solutions for u(t) that permit relatively high values of d(t) or u(t) at any given time. to find the optimal control, u * (t), that minimizes j(u), we follow standard results from optimal control theory applied to systems of ordinary differential . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint equations [ ] . these techniques make use of pontryagin's maximum principle to determine the pointwise minimum of the hamiltonian of the system, h, using adjoint variables, λ, that correspond to each of the state variables. to solve for the adjoint variables, we first define the hamiltonian, we then define differential equations describing the behavior of each adjoint variable as the negative of the partial derivative of h with respect to the state variable corresponding to each adjoint variable. this yields where eqn. ( ) can be solved backwards in time with transversality conditions at time t equal to zero for each adjoint variable. to find the pointwise optimal control, u * , we find the value of u that minimizes ∂h ∂u , which yields . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . the optimal control is subject to a lower bound of and an upper bound of u max , with those values used for u * whenever the right hand side of eq. ( ) yields values outside those bounds. to find u * (t) numerically, we use the forward-backward sweep method [ ] , which involves first solving for the state variables forward in time, next solving for the adjoint variables backward in time, and then plugging the solutions for the relevant state and adjoint variables into eqn. ( ) , subject to bounds on u(t). as is often the case for optimal control problems [ ] , we found that we needed to perform these steps iteratively and with a convex combination of controls across iterations to achieve convergence. specifically, we repeated the forward-backward sweep process times, after which we took newly proposed solutions of u * (t) as the average of the most recent solutions, until the algorithm was stopped after , iterations. we assessed convergence with a statistic defined as where smaller values indicate better convergence. all numerical solutions were obtained with a runge-kutta routine implemented with the ode function from the desolve package [ ] in r. in table , we specify low, intermediate, and high values of each parameter. for some parameters, estimates matching our parameter definitions were taken from other studies. for other parameters, additional steps were necessary to match our parameter definitions. we elaborate on the latter below. transmission coefficient, β. we based values of this parameter on assumptions about r by solving for β as a function of r and other parameters in eqn. ( ) . because estimates of r for covid- vary widely [ ] , we chose values that span a range of estimates that may be applicable to the us. background birth and death rates, µ and δ. we parameterized µ consistent with a rate of , , births in a population of million in the us in [ ] . to achieve a constant population size in the absence of covid- , we set δ equal to µ. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint probability of death among hospitalized cases, ∆ and h. we assume that ∆ − is equal to early estimates from the us ( . %) [ ] and that ∆ + is equal to estimates from italy ( . %) [ ] . estimates of how quickly ∆ + might be approached have not been made empirically, so the value of h was assumed. the intermediate value of h = corresponds to an increase of h of % beyond h max resulting in % of the maximum increase from ∆ − to ∆ + . progression through hospitalization, η. we used line-list data [ ] to estimate a mean ( . days) and standard deviation ( . days) of the time between hospital admission and either discharge or death. timing of vaccine introduction, τ ν . experts have stated that a vaccine against covid- could be available to the public by spring [ ] . we used april , as our default value for this parameter. vaccination rate, ν. in the h n pandemic, million doses of vaccine were administered between october and april [ , ] . consistent with that, our default value of ν resulted in . % of the population being vaccinated each day. hospital capacity, h max . we based our definition of this parameter on hospital beds and did not include other aspects of hospital resources, such as icu beds, ventilators, or hospital staffing. specifically, we adopted an estimate of , beds available for covid- patients in the us by the institute for health metrics and evaluation [ ] . maximum effect of control, u max . a model incorporating survey data and agebased contact patterns estimated that social distancing could reduce transmission of sars-cov- by % [ ] . based on this, we used . as an intermediate value of this parameter and . and . as lower and upper values. to obtain realistic behavior of the model, we calibrated it to match the cumulative number of reported deaths in the us within the first days of (i.e., by april ), which we obtained from the new york times [ ] . we focused our calibration on the parameter ι, due to the difficulty of empirically estimating the rate at which imported infections appear. to obtain a value of ι that resulted in the model matching the reported number of deaths, we simulated the model across values of ι evenly spaced between − and − on a log scale, performed a linear interpolation of the simulated number of deaths across those values of ι, and found the value of ι that most closely matched reported deaths. we calibrated the model under a total of different parameter scenarios, crossing low, intermediate, and high values of r with low, intermediate, and . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . high values of u max and low and high values of ω. the latter represents the proportion of all deaths caused by covid- that were reported. because npis began going into effect in the us within the timeframe of this calibration period, we calibrated the model subject to an assumed pattern of u(t) through the first days of . we chose a logistic functional form for this, with a minimum of and a maximum of u max . two parameters that control the midpoint and slope of the increase from to u max were selected by an informal process of trial and error, with the goal of having the model's predictions of deaths over time match the timing of reported deaths under all parameter scenarios. we also used this process to select the date on which importations were initiated through ι. under each of the different scenarios we considered about r , u max , and ω, our model was able to reproduce the numbers and timing of reported deaths in the us reasonably well (figure ) . different values of ι were required to do so under different values of those other three parameters, but all values of ι ranged − - − ( table ) . taking into account the population of the us, this equates to a range of - imported infections per day. higher values of r resulted in lower values of ι, given that fewer importations were required to generate the reported numbers of deaths when there was more local transmission. for similar reasons, ι was lower when u max was lower. the value of ω had a negligible effect on ι. in addition to ι, we also calibrated the date on which importations began (february ) and u(t) during the first days of . even though the first imported cases in the us began appearing before february [ ] , it is . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint not surprising that our model better matched the data with a slightly later start date, given that the model did not allow for increases in importation over time, as likely occurred [ ] . the calibration of u(t) was consistent with u(t) starting at zero, reaching half of u max on april , , and increasing by a maximum of . u max per day around that time. the timing of these changes is approximately two weeks later than changes in google mobility data from the us [ ], which may be a consequence of our model moving some individuals from exposure to death too quickly given that residence time in each compartment is exponentially distributed. in general, we do not interpret any of the calibrated parameter values as reliable estimates of empirical quantities. such an interpretation would require analyses that more carefully account for data-generating processes and sources of uncertainty. rather, our objective in this calibration exercise was to ensure that the model's behavior is reasonably consistent with empirical data, which we feel was accomplished. for our main results, we obtained solutions to u * (t) under a total of parameter combinations, crossing the parameter sets in table with seven values of c spanning − to − by factors of ten. in one representative example (figure , left), we observed that solutions of u * (t) converged to a set of similar solutions after several hundred iterations. as expected, the objective functional decreased during those initial iterations and remained low thereafter . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . ( figure , right). to assess convergence by the statistic in eqn. ( ) , we selected the iterations with the ten lowest values of j(u) in the last iterations. of parameter combinations, % had final solutions of u * (t) with convergence statistics below − , % below − , and all below − . comparing values of the two components of the objective functional, t t d(t) dt and t t u(t) dt, across different parameter values provides insight into the range of behavior of the model and its response to control. because they are similar to the components of the objective functional but more easily interpretable, we describe effects of model parameters on t t d(t)dt (cumulative deaths) and t t u(t)dt (cumulative time under control). the parameter c, which controls the weight of the two terms in j(u), led to a transition from minimizing cumulative deaths to minimizing cumulative time spent under control as it varied from − to − ( figure ). the parameters r and u max both influenced the extent to which cumulative deaths could be minimized. with a high value of u max , deaths could be kept relatively low across all values of r explored, provided that c ≤ − . with a low value of u max and a high value of r , cumulative deaths could only be reduced by around % as c varied across its entire range from − to − . the parameter ω had essentially no influence on the components of the objective functional ( figure ). we first consider the scenario where r = and u max = . , because those are the conditions under which control has the greatest potential to influence . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . the pandemic. under c = − , u * (t) remains at u max until late june , after which it settles down to around % of u max until late ( figure ). this results in hospitalizations dropping from their peak in april and remaining very low through . the susceptible population remains very high and only begins eroding once a vaccine is introduced. with a higher value of c = − , u * (t) drops to around % of u max in may ( figure ). as a result, hospitalizations rebound and exceed hospital capacity by around a third in june and july before falling again, after u * (t) returns to u max . from august onward, a steady decline in u * (t) allows hospitalizations to be maintained at moderate levels before rebounding and exceeding hospital capacity once again for several months in . with the highest value of c = − , u * (t) drops almost to zero at the beginning of may ( figure. ) . this results in a rapid increase in hospitalizations, which is followed by an increase in u * (t). by the end of june, the susceptible population has been depleted to the point that herd immunity begins to obviate the need for control and u * (t) declines to zero by october . during this large second wave in summer , hospital capacity is ex-. cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint fig. optimal control under parameters with maximal ability to control the pandemic, with minimal weighting on minimization of deaths. panels show the optimal control (bottom) and its impacts on the dynamics of hospitalized (middle) and susceptible (top) compartments with r = , umax = . , ω = . , and c = − . vaccination is introduced at the time indicated by the arrow, with the vaccinated population (orange) reducing the susceptible population. through april (gray shading), the value of the control is fixed according to its calibrated trajectory. the dashed horizontal line indicates hospital capacity. ceeded by more than -fold. this results in cumulative deaths equaling % of the population, which is approximately one order of magnitude greater than when c = − and two orders of magnitude greater than when c = − . under other conditions about the transmissibility of the virus and the potential for npis to reduce transmission, control is less capable of curtailing the epidemic. with r = . and u max = . , hospitalizations peak in early summer at twice hospital capacity and take until december to fall below hospital capacity, even with the most aggressive value of c that we considered ( − ) (figure ). with r = , u max = . , and c = − , hospitalizations peak in summer at thirty times hospital capacity (figure ). this results in extensive herd immunity and the relaxation of control in , albeit at the cost of extensive deaths (figure ) . optimal controls under a wider variety of parameter combinations can be explored interactively at http://covid optimalcontrol.crc.nd.edu. our calibration procedure resulted in a single assumption about the trajectory of u(t) prior to april , . control during this period was fixed in analyses in section . , with the flexibility to define u * (t) only allowed during the period from may , to december , . here, we explore how npis . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint fig. optimal control under parameters with maximal ability to control the pandemic, with maximal weighting on minimization of deaths. panels show the optimal control (bottom) and its impacts on the dynamics of hospitalized (middle) and susceptible (top) compartments with r = . , umax = . , ω = . , and c = − . vaccination is introduced at the time indicated by the arrow, with the vaccinated population (orange) reducing the susceptible population. through april (gray shading), the value of the control is fixed according to its calibrated trajectory. the dashed horizontal line indicates hospital capacity. fig. optimal control under parameters with maximal ability to control the pandemic, with maximal weighting on minimization of deaths. panels show the optimal control (bottom) and its impacts on the dynamics of hospitalized (middle) and susceptible (top) compartments with r = , umax = . , ω = . , and c = − . vaccination is introduced at the time indicated by the arrow, with the vaccinated population (orange) reducing the susceptible population. through april (gray shading), the value of the control is fixed according to its calibrated trajectory. the dashed horizontal line indicates hospital capacity. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint initiated one to three weeks earlier or later in spring would affect optimal controls in the period after. we focus this analysis on scenarios in which c = − , which results in the optimal control problem seeking to minimize deaths as aggressively as any scenario that we explored. consequently, we interpret changes in the optimal control in this section to reflect changes in constraints on what solutions of u * (t) are possible, rather than changes in the balance between d(t) and u(t) in the optimization. across all combinations of r , u max , and ω that we considered, cumulative deaths through and decrease when control begins earlier and increase when control begins later (figure , red) . in the scenario in which a delay in the initiation of control has the smallest effect (r = , u max = . ), cumulative deaths increase by % with a three-week delay. in the scenario in which a delay in the initiation of control has the largest effect (r = , u max = . ), cumulative deaths increase -fold with a three-week delay. the overall amount of time spent under control throughout and increases when control begins earlier and decreases when control begins later ( figure , blue). this is the case across all combinations of r , u max , and ω that we considered. in part, this owes to less time spent under control through april , , when u(t) is fixed and not subject to optimization. in addition though, delays in the initiation of control result in a higher prevalence of infection by the beginning of the optimization period, which results in higher levels of subsequent transmission, greater depletion of the susceptible population, and less need for control later in the period of optimization (compare figure with figure ). under our model, the ability of npis to minimize deaths depends to a large extent on the maximum effect that npis could have on transmission, as captured by the parameter u max . if u max is high ( . ), our model predicts that maintaining transmission at a low enough level that hospital capacity will not be exceeded could be possible. if u max is low ( . ), our model predicts that there may be limited scope to curtail the pandemic, even if control is sustained for a long period of time. if u max is intermediate ( . ), our model predicts that the potential impact of control is sensitive to the value of r . these results emphasize the importance of careful estimation of these parameters as the pandemic progresses [ ] . this includes accounting for geographic differences in both u max and r [ , ] . the balance between minimizing deaths versus days under control is determined by the parameter c in our model, which mirrors the weighting between these factors that government leaders will need to consider as they make decisions in coming months. in the event that npis are effective, our analysis shows that they will need to be sustained at a high level until sometime in summer , at which time they can be relaxed to a small degree but will still need to be maintained at a relatively high level thereafter. scenarios that . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . place greater weight on minimizing days under control provide insight into the possible consequences of relaxing npis prematurely. in a scenario in which the effect of npis is reduced moderately in may, resuming npis at high levels soon after becomes necessary to react to a second wave later in the summer. in a scenario in which npis are reduced more drastically in may, an extremely large second wave occurs in summer that exceeds hospital capacity many times over. our conclusion that prolonged control is needed to avoid a resurgence that would greatly exceed healthcare capacity is in line with results from other modeling studies [ , , , , ] . some differ though in that they consider scenarios in which control is implemented intermittently, with periods of relaxed control in between periods of maximal control [ , , ] . in the limited number of direct comparisons of intermittent and continuous strategies that have been performed for covid- to date, continuous strategies appear to be capable of more effectively limiting transmission to low levels [ , ] . the sustainability of either strategy would benefit from the ability to transition . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . away from heightened social distancing and more toward diagnostic testing, contact tracing, and case isolation [ , ] . our results tend to agree with those from other optimal control analyses of covid- , although our analysis goes beyond those studies in some ways. in general, ours and other studies share the conclusion that heightened control early in the pandemic is important for achieving long-term success. like our analysis, some anticipate that partial relaxation of controls may be possible over time [ , ] , whereas others focus on strategies intended to have a more limited duration to begin with [ , , ] . importantly though, our analysis goes further than others in exploring sensitivity of the optimal control to model parameters. specifically, we show that preventing a large wave that overwhelms health systems may not even be possible under some parameter combinations (low u max , high r ) and that prioritizing the minimization of deaths versus days under control leads to vastly different outcomes. we also constrain levels of control applied through april , making the optimization more relevant to future decision making. shifting the timing of the initiation of control shows that constraints about past levels of control strongly affect future possibilities for the extent to which deaths can be minimized and the level of control required to achieve that, consistent with other results [ ] . the goal of our analysis is to provide qualitative insights into the implications of alternative approaches to control, rather than to make quantitative predictions about future events. some key limitations of using our model for . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint the latter purpose include our omission of subnational variation in epidemic dynamics [ , ] , differentiation among alternative npis [ ] , and age differences in contact patterns [ ] and risk of hospitalization [ ] . at least with respect to the possibility of different levels of control for different age groups, one study found that age-specific optimal controls were all relatively similar for npis against pandemic influenza [ ] . additional limitations that affect our model's suitability for making future predictions include its deterministic nature and the rudimentary calibration procedure that we performed, which was sufficient to provide a basis for qualitative analyses but that would need refinement for application of our model to inference or forecasting. in conclusion, our analysis suggests that we are at a critical juncture in the pandemic, when decisions about the continuation or relaxation of npi-based control strategies could have major implications for the possibility of keeping transmission below levels that health systems can cope with. at the same time, our analysis highlights the role that constraints play in determining optimal levels of control going forward, both in terms of constraints on epidemiological parameters and on levels of control prior to the time that a decision is made about future actions. going forward, reducing transmission in the near term would give decision makers greater flexibility in the range of decisions available to them in the future, and gathering high-quality data could help reduce uncertainty about the consequences of those decisions. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint ncov data working group: epidemiological data from the ncov- outbreak: early descriptions from publicly available data optimal control strategies for dengue transmission in pakistan non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base cnn: lead scientist says coronavirus vaccine could be ready soon covid- : towards controlling of a pandemic backward bifurcation and optimal control in transmission dynamics of west nile virus h n flu vaccine h n pandemic timeline control and prevention: severe outcomes among patients with coronavirus disease (covid- ) -united states the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak optimal tuberculosis prevention and control strategy from a mathematical model based on real data impact assessment of non-pharmaceutical interventions against coronavirus disease and influenza in hong kong: an observational study the effect of nonpharmaceutical interventions on covid- cases, deaths and demand for hospital services in the uk: a modeling study optimal covid- epidemic control until vaccine deployment. medrxiv further notes on the basic reproduction number editorial: covid- in the usa: a question of time report -impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand report -estimating the number of infections and the impact of nonpharmaceutical interventions on covid- preparedness and vulnerability of african countries against importations of covid- : a modelling study estimation of country-level basic reproductive ratios for novel coronavirus (covid- ) using synthetic contact matrices. medrxiv first case of novel coronavirus in the united states quantifying the impact of physical distance measures on the transmission of covid- in the uk 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characteristics of patients dying in relation to covid- in italy reconciling early-outbreak estimates of the basic reproductive number and its uncertainty: framework and applications to the novel coronavirus (sars-cov- ) outbreak. medrxiv optimal timing for social distancing during an epidemic. medrxiv ( ) estimating unobserved sars-cov- infections in the united states heterogeneous local dynamics revealed by classification analysis of spatially disaggregated time series data optimal covid- quarantine and testing policies. eief working papers series the effect of control strategies to reduce social mixing on outcomes of the covid- epidemic in wuhan, china: a modelling study control strategies to curtail transmission of covid- . medrxiv optimal strategies of social distancing and vaccination against seasonal influenza solving differential equations in r: package desolve optimal control and sensitivity analysis of an influenza model with treatment and vaccination forecasting covid- impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next months mathematical modelling of covid- transmission and mitigation strategies in the population of ontario, canada time-variant strategies for optimizing the performance of nonpharmaceutical interventions (npis) in protecting lives and livelihoods during the covid- pandemic acknowledgements thanks to the university of notre dame's center for research computing for computing resources. key: cord- - m ozm authors: wang, huijie; li, na; huang, huaqiong title: asthma in pregnancy: pathophysiology, diagnosis, whole-course management, and medication safety date: - - journal: can respir j doi: . / / sha: doc_id: cord_uid: m ozm asthma in pregnancy is a health issue of great concern. physiological changes and drug compliance during pregnancy can affect asthma control in varying degrees, and the control level of asthma and the side effects of asthma medications are closely related to the adverse perinatal outcomes of mother and fetus. this article provides an update on the available literature regarding the alleviating or aggravating mechanism of asthma in pregnancy, diagnosis, disease assessment, and systematic management, to provide a new guidance for physician, obstetric joint doctor, and health care practitioner. asthma in pregnancy is a common respiratory disease, and the burden of it has gradually increased worldwide, which has become one of the most common public health problems. e incidence of asthma in pregnancy increases from % to % in the united states after [ ] and % of women suffer from asthma during pregnancy in britain [ ] and % in australia [ ] . asthma control levels often change during pregnancy. it is generally believed that one-third of asthma patients are aggravated due to pregnancy, and most occur in the middle of pregnancy; one-third improved, and no significant changes are observed in the remaining / of patients. but a latest multicase-control study shows that the percentage of asthma worsening during pregnancy is . %, lower than the previous data, and the worsening is significantly associated with the severity of the disease [ ] . ere are also many problems in the control of asthma during pregnancy. studies show that about % of patients have poor control of asthma during pregnancy, inhaler technology is not correct in . % of cases, only % of patients know the difference between asthma reliever and controlled medications, . % of patients receive a written asthma action plan, % of patients have spirometry in the past years, and . % of them have peak expiratory flow meter at home [ ] . studies have shown that maternal asthma increases the risk for adverse complications in fetuses and mothers, including sga (small for gestational age), lbw (low birth weight), congenital malformations (cleft lip or cleft palate), increased perinatal mortality, pb (premature birth), maternal preeclampsia, gestational hypertension, gestational diabetes, prenatal hemorrhage, caesarean section, urinary tract infection, excessive amniotic fluid, and premature rupture of membranes, especially for those patients with severe or uncontrolled asthma during pregnancy [ , ] . onset during pregnancy e pathogenesis of asthma remission or aggravation during pregnancy is related to the physiological or pathological changes caused by pregnancy, mainly including the mechanical changes caused by uterine enlargement and the direct or indirect effects of hormonal changes during pregnancy. with the increase of uterus and abdominal pressure, the diaphragm is elevated by - cm, subcostal angle increased % ( °to °from early to late pregnancy), and the transverse and anteroposterior diameter of thoracic increased. e above changes are partially compensated by relaxation of ligamentous attachments of the ribs which leads to the decrease of the thoracic compliance. as a result, the total lung volume decreases by % and frc (functional residual capacity) decreased by % [ ] . moreover, the increased body weight leads to larger neck circumference and smaller oropharyngeal area which contributes to dyspnea during pregnancy [ ] . during pregnancy, in order to meet the needs of maternal and fetal metabolism, a series of important changes occur in hormone levels, including the obvious increase of progesterone, estrogen, cortisol, and prostaglandin, all of which have different effects on the course of asthma. progesterone is a stimulant of respiratory dynamics, which can increase the sensitivity of respiratory center to carbon dioxide, while estrogen can increase the sensitivity of progesterone receptor in respiratory center and jointly participate in the change of respiratory function [ ] . e minute ventilation increases by %- % which is mainly due to a % increase in tidal volume, while there is no significant change in respiratory rate. tlc (total lung capacity), vc (vital capacity), lung compliance, and dlco (diffusion capacity) remain unchanged. fvc (forced vital capacity), fev (forced expiratory volume in s), the ratio of fev to fvc, and pefr (peak expiratory flow rate) have no significant changes during pregnancy compared with nonpregnancy [ ] . erefore, spirometry can be used to identify dyspnea in normal pregnancy and reflect the changes in respiratory diseases. in addition to acting on the respiratory center, progesterone can mediate mucosal vasodilation and congestion, resulting in the increase of pregnancy rhinitis and epistaxis incidence [ ] and oropharyngeal and laryngopharyngeal airways that contribute to the attack of asthma in pregnancy. estradiol can increase maternal innate immunity and cell-or humoral-mediated adaptive immunity. low concentration of estradiol can promote cd + cell response and cell-mediated immunity. high concentration of estradiol can enhance cd + cell response and humoral immunity. progesterone inhibits the maternal immune response and changes the balance between and responses. although cell-mediated immunity is more important in respiratory viral infections, the transfer of to immunity is considered to be an important mechanism for asthma induced by hormones during pregnancy [ , ] . women are in the state of hypercortisonism during pregnancy; meanwhile, the placenta secretes both crh (corticotropin-releasing hormone) and acth (adrenocorticotropic hormone), which results in the increase of free cortisol and conjugated cortisol during pregnancy. e increased free cortisol mediates the increase of beta-adrenoceptor and the enhancement of bronchiectasis [ ] . increased secretion of prostaglandin e (pge ) during pregnancy, through anti-inflammatory, inhibition of smooth muscle cell proliferation, bronchial relaxation, and other mechanisms, has a protective effect on the incidence of asthma. in addition, progesterone also has the effect of changing the airway smooth muscle tension and causing bronchiectasis [ ] . ese factors are associated with asthma remission during pregnancy. generally speaking, the effects of mechanical and biochemical changes on the respiratory system of pregnant are very complex, especially the effects of various hormones on the respiratory center, peripheral airway, and immune system, leading to nonasthmatic pregnant women experiencing varying degrees of dyspnea during pregnancy. for pregnant women with asthma, it is very important to strengthen the management of asthma during pregnancy to avoid maternal hypoxia and maintain adequate fetal oxygenation. general asthma is defined by the history of more than one type of respiratory symptoms such as wheeze, shortness of breath, chest tightness, and cough that vary over time and in intensity, often appear or worsen with viral infections, and attack at night or on waking, usually triggered by exercise, laughter, allergens, and cold air, together with variable expiratory airflow limitation [ ] . if one of the tests is positive including bronchodilator reversibility test, bronchial provocation tests, and pef variability, it can confirm variable expiratory airflow limitation. compared with general asthma, asthma in pregnancy has similar clinical manifestations. however, if a pregnant woman only complains of shortness of breath or chest tightness, we must be careful to diagnose based on her medical history. as we know, over two-thirds of pregnant women experience some form of shortness of breath or chest tightness during the gestation period because of physiological changes of pregnancy [ ] . in addition, it would not be advisable to carry out a bronchial provocation test to prevent maternal hypoxia and fetal distress. assessment of asthma usually includes asthma control (both symptom control and future risk of adverse outcomes), treatment issues particularly inhaler technique and adherence, and any comorbidities that can contribute to symptom burden and poor quality of life [ ] . lung function, feno (exhaled nitric oxide), act (asthma control test) scores [ ] , and blood eosinophil counts are important tools for asthma assessment. pefr is used to monitor lung function, which should be to l/min, but the personal best value is required. maintaining - % of personal best value is considered normal range for asthma patients. feno is an indicator of airway inflammation; studies have found that pregnant women with asthma have the same feno as before, which is associated with asthma control [ ] [ ] [ ] . adjusting treatment of asthma in pregnancy according to feno can reduce acute attacks and neonatal admission rate. e assessment of asthma in pregnancy is a collaborative effort by physician, obstetric joint doctor, and health care practitioner. it is necessary not only to evaluate pregnant women but also to comment on the growth and development of the fetus. and the frequency of assessments needs to be more frequent, once a month is recommended by gina (global initiative for asthma). whole-course management of asthma in pregnancy can reduce the negative effects of fluctuations in asthma symptoms or acute exacerbation on pregnant women and the fetus. e gina guidelines suggest that poor asthma control and acute exacerbations during pregnancy are more risky than taking asthma medications. e long-term goals of asthma management are to achieve good symptom control, maintain normal activity levels, and minimize the risk of acute attacks, irreversible damage to lung function, and drug-related adverse effects. for asthma in pregnancy, it is also essential to avoid side effects of drugs on pregnant and fetuses and give birth to healthy babies. e principle of asthma treatment is defined as the selection of appropriate treatment based on the severity and control level of patient. a written asthma control plan should be developed for each newly diagnosed patient, regular follow-up and monitoring, and adjusting treatment according to patient control level to achieve and maintain asthma control. ough the principles of medication for asthma in pregnancy are similar to those of nonpregnant patients, there are differences between them in the implementation of stepwise approach. for general asthma, the control level of asthma is assessed at months after the use of controller medication. ose with good asthma control are treated with step down treatment, while those with persistent asthma symptoms or acute exacerbation are treated with step-up treatment. for asthma in pregnancy, the gina guidelines and the australian asthma handbook recommend that asthma control levels should be assessed monthly. step-down treatment is not a priority in order to avoid acute exacerbation of asthma unless the pregnant woman's drug dosage is inappropriate [ , ] . in addition, anti-ige monoclonal antibodies and specific immunotherapy should not be initiated during pregnancy [ ] . currently, the most commonly used and safe drugs during pregnancy include glucocorticoids, beta -agonist, anticholinergics, theophylline, leukotriene receptor antagonists (ltras), omalizumab, and allergen immunotherapy (ait). among glucocorticoids, inhalation administration is predominant. inhaled glucocorticoids (ics) can effectively inhibit the number and activity of inflammatory cells in the airway; moreover, it can significantly reduce the side effects of systemic medication since its effect on local airway. budesonide is the most commonly used and safe ics during pregnancy. a study based on danish medical registries, of primiparous women who gave birth to a live-born singleton in - , has found no association between use of glucocorticoids (both ics and oral glucocorticoids, ocs) in early pregnancy and risk of oral clefts or congenital malformations overall in the offspring [ ] . tegethoff et al. have studied mother-child pairs from the danish national birth cohort and found that ics for asthma treatment during pregnancy (n � ; . % budesonide, . % fluticasone, . % beclomethasone, and . % other or unspecified glucocorticoids) is not associated with offspring disease risk in most categories, except for offspring endocrine, metabolic, and nutritional disorders [ ] . however, mortenet al. have indicated that fraction of exhaled nitric oxide-(feno-) guided asthma management during pregnancy reduces doctor-diagnosed asthma in the offspring at the stage of preschool, partly mediated through alterations in use and dosing of ics during the trial of managing asthma in pregnancy [ ] . many studies have failed to clarify whether the use of ics increases the risk of teratogenic, but it is clear that asthma patients who do not use ics have a significantly increased risk of giving birth to low-weight children, suggesting that the risk of poor asthma control is much higher than medication [ ] . vicki et al. support this conclusion. eir research suggests that the placenta plays an important role in regulating fetal growth by producing an enzyme, βhydroxysteroid dehydrogenase- ( β-hsd- ), which can convert endogenous and exogenous glucocorticoids derived from the maternal circulation to their inactive metabolites. as a consequence, β-hsd- activity has a positive correlation with birth weight. asthmatic patients without using ics and β-hsd- activity are reduced in placentae of female fetuses and that may be associated with inflammatory factors produced during the pathogenesis of asthma. while using low, moderate, or high doses ics will not affect placental capacity to metabolise glucocorticoids, so it does not affect the birth weight, which may be related to the fact that ics is able to increase production of placenta β-hsd- . additionally, uncontrolled asthma frequently shows associations with adverse perinatal outcomes, as previously demonstrated with exacerbations in cohorts of asthma in pregnancy, and the continued use of ics with budesonide is recommended and has a particularly good safety profile [ ] . in short, asthma itself has more adverse effects on the placenta than its treatment [ ] . in addition, studies have shown that low doses of ics in patients with asthma during pregnancy can reduce the risk of acute exacerbation and readmission, but there are safety issues with high-dose ics. lucie blais et al. have conducted a cohort study of pregnancies of women with asthma ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and found that women who use > μg/day ics are significantly more likely to have a baby with a malformation than the women who use > to μg/day, while women who use > to μg/day are not found to be more at risk than women who do not use icss during the first trimester [ ] . a cohort study incorporating pregnancies also discovers no increased prevalence of lbw, pb, and sga in pregnant women with asthma for longacting beta -agonists (laba) use and ics doses < μg/ day, while a trend for an increased prevalence of the outcomes is seen for ics doses above μg/day. maternal glucocorticoid-regulated systems are susceptible to ics canadian respiratory journal ( μg/day for first and second trimester and μg/day for third trimester) for asthma during pregnancy, but glucocorticoid-regulated pathways in the fetus are not affected by ics use, which implied that ics use for the control of asthma during pregnancy is unlikely to contribute to adverse effects on fetal growth and development [ ] . ocs is sometimes necessary depending on the severity of asthma. but we should keep in mind that, among all ocs, prednisone, prednisolone, and methylprednisolone can cross the placenta at very low concentrations, while dexamethasone and betamethasone reach the fetus at higher concentrations. among them, prednisone is the most common ocs. before entering the fetal blood circulation through the placenta, % of blood concentration is inactivated by the action of β-hsd- , with little impact on the fetus. accumulating evidence suggests that the use of ocs can increase the incidence of cleft lip and palate in the fetus, especially in the first trimester of pregnancy. e incidence of cleft lip and palate in general population is . %, while in oral glucocorticoids-taking pregnant women it is . % [ ] . however, there is also study which believed that the risk of cleft palate is uncorrelated with the application of ocs during pregnancy [ ] . and using ocs throughout pregnancy may increase the incidence of preeclampsia, preterm birth, and low birth weight [ ] . an animal experiment shows that glucocorticoid exposure in early placentation could contribute to preeclampsia, with the mechanisms involving inhibition of trophoblast proliferation, migration, invasion, and epithelial-mesenchymal transition by glucocorticoid [ ] . in population-based studies, ocs has also been reported to increase the risk of preeclampsia [ ] . jennifer et al. have conducted a meta-analysis on papers from to which are related to the risk of adverse outcomes in neonates with acute attack of asthma, oral corticosteroid, or asthma severity, which shows that acute attack of maternal asthma and oral corticosteroid use have a significant effect on low birth weight and preterm delivery; moderate-to-severe asthma during pregnancy is also associated with an increased risk of sga and low birth weight infants [ ] . ere is evidence that asthma in pregnancy perceives a higher risk of ocs medication use on the fetus, compared with ics treatment, which may lead to abnormal behavior of women during pregnancy [ ] . in terms of the risk tradeoffs between poor asthma control and the use of ocs, the current consensus remaining is that the risk of inadequate treatment is higher than the potential risk of systemic glucocorticoids. beta -agonists are suitable for asthma patients of all levels during pregnancy, and quantitative inhalation or atomization of solution is the main administration method. short-acting beta -agonist (saba) including salbutamol, terbutaline, and pirbuterol is used as reliever medications. naepp recommended that the preferred choice in saba is salbutamol because of its safety data for pregnant with asthma. laba, salmeterol, and formoterol are as controlled medications. laba is similar to salbutamol in pharmacologic and toxicologic profile; while there are limited data on their use during pregnancy, salmeterol might be chosen which has been available longer [ ] . report indicates that using of long-acting beta -agonists (laba) during pregnancy will not increase the prevalence of the perinatal outcomes [ ] . however, in the current studies, the relationship between beta -agonists and perinatal adverse outcomes is still controversial. a research finds no significant relationships between major congenital malformations and sga infants with exposure to beta -agonists, theophylline, cromolyn, and corticosteroids during the first trimester or at any time [ ] . a retrospective cohort study of pregnancies finds no increased risk of congenital malformations with saba exposure during the first trimester, while using laba in the first trimester is significantly associated with a higher risk of major "cardiac malformations" and major "other and unspecified congenital malformations." however, bias due to different asthma severity or chance should be considered [ ] . a review identified original studies; studies report that use of saba during pregnancy increases risk of congenital malformations, while one study reports that high doses of saba decrease the risk. four studies indicate that beta -agonists (saba and/or laba) contribute to increased risk of congenital malformations; one study shows a significant increased risk with laba. one study shows that laba led to low birth weight babies [ ] . a populationbased case-malformed control study including , registrations of congenital anomalies from euromedicat registries finds that exposure to first trimester with inhaled beta -agonists increases the risk of cleft palate and gastroschisis. also, exposure to the combination of laba and ics is associated with renal dysplasia [ ] . however, in the cohort study of cases of hypertensive disorders of pregnancy and cases of preeclampsia/eclampsia, laba use is not associated with increased risks of hdp or preeclampsia/eclampsia, suggesting the safety of labas for the cure of asthma in pregnancy [ ] . besides, maternal asthma is closely associated with increased risk of offspring autism spectrum disorder (asd), but this adverse effect is not associated with prenatal exposure to asthma medications including laba [ ] . gina ( ) suggests that saba alone is no longer as the recommended therapeutic strategy for asthma; instead gina recommends that all adults and adolescents with asthma should receive either symptomdriven (for mild asthma) or daily inhaled anti-inflammatory controller treatment, to reduce their risk of serious exacerbations and to control symptoms. anticholinergics include the shortacting muscarinic antagonist (sama), ipratropium bromide, and long-acting muscarinic antagonists (lamas), tiotropium bromide. gina guidelines suggest that combination of formoterol and saba could be used to treat severe acute asthma attacks. previous studies also have demonstrated that the use of saba plus ipratropium bromide can effectively improve severe acute asthma control. however, there is still much controversy over whether ipratropium bromide can reduce the admission rate of patients and improve their lung function [ ] . a study finds that, for patients with poor asthma control by applying ics or ics combined with laba, tiotropium as add-on therapy can improve pulmonary function and reduce the frequency of exacerbations [ ] . in a systematic review of randomized-placebo controlled trials, the use of anticholinergic tiotropium ameliorates asthma control in patients with moderate symptomatic asthma who have already received medium-to-high doses ics or ics/laba [ ] , while there are few studies on the use of anticholinergics in asthma patients during pregnancy. more studies are needed to confirm the relationship between anticholinergic drugs and perinatal adverse outcomes. low-dose theophylline is considered as an alternative for mild persistent asthma during pregnancy and as an alternative adjunctive long-acting bronchodilator therapy for moderate-to-severe asthma when ics alone cannot provide adequate control of patient's asthma [ ] . eophylline can pass through the placental barrier. ere is no significant difference in theophylline concentration between maternal and umbilical cord serum, when the levels of theophylline are greater than μg/ml, transient neonatal vomiting, tremor, and tachycardia can occur [ ] . e blood concentration should be maintained at - μg/ ml in nonpregnant asthma patients and - μg/ml in pregnant women [ ] . when serum theophylline is higher than μg/ml, the risk of theophylline toxic reactions will increase [ ] . since the recommended dose of theophylline is close to the toxic dose, the plasma or urine concentration must be closely monitored during the treatment, especially for pregnant women whose liver metabolic function decreased. e updated gina guidelines indicate that lowdose theophylline (serum concentrations must be monitored closely) is considered as an alternative, but not a preferred treatment for mild persistent asthma during pregnancy. for moderate-to-severe asthma during pregnancy, theophylline may be considered as an alternative adjunctive long-acting bronchodilator therapy but not a preferred therapy when ics alone does not provide adequately asthma control during pregnancy. additionally, theophylline use has no or minimal effects on fetal growth and reduces perinatal complications when maternal asthma is adequately controlled [ ] . in a multicenter prospective study, compared with the effects of ics and oral theophylline on asthmatic pregnant women, the two cohorts have similar rates of asthma exacerbations, asthma outcomes, and obstetric and perinatal outcomes, while theophylline group has more adverse events, drug withdrawal rate during observation period and fev < % predicted value of lung function than glucocorticoids group [ ] . at present, the use of controlled-release theophylline preparations is advocated, which can dilate bronchus for - hours, and is conducive to the control of night asthma. intravenous aminophylline is mostly used in acute asthma attacks, and no teratogenic effect has been found. antileukotrienes are divided into two categories: ltras including zafirlukast and montelukast and -lipoxygenase pathway inhibitors, zileuton. among them, zileuton is not recommended for use in pregnancy because of the lack of relevant research and the need to monitor liver function during medication. ltras alone are less effective compared with ics and they are generally taken in combination with other asthma medications. however, the use of ltras in combination with ics has the same effect as those of labas in combination with ics in steroid-naïve asthmatic patients [ ] . with the exposure to ltras in asthma patients during pregnancy, no increased risk for major birth defects is observed in a cohort study of first-trimester-exposed pregnancies [ ] . and adverse outcomes including pregnancy loss, gestational diabetes, preeclampsia, low maternal weight gain, preterm delivery, low apgar scores, or small size are discovered in a cohort study of infants treated with leukotriene receptor antagonists in pregnancy [ ] . e ltras are only recommended for patients who had a favorable response to them before becoming pregnant. ey are an alternative to icss and are not preferred as a treatment option in mild persistent asthmatics during pregnancy [ , ] . anti-ige monoclonal antibody omalizumab is as an add-on therapy for the treatment of nonpregnant patients with moderate-to-severe persistent asthma that is inadequately controlled with ics and has the effect of preventing exacerbation, reducing the frequency of asthmatic symptoms, reducing the frequency of emergency room visit or hospital admission, and reducing the steroid dose. a prospective, observational study, expect (the omalizumab pregnancy registry), includes pregnant women who exposed to ≥ dose of omalizumab within weeks prior to conception or at any time during pregnancy and collected data on pregnant women at the time of data cut-off. e proportions of major congenital anomalies ( . %), prematurity ( . ), low birth weight ( . %), and sga ( . %) observed in the expect registry are consistent with findings from other studies in this asthma population. e prevalence of major congenital defects in expect is no higher than those reported in the general population with asthma. omalizumab does not appear to increase the risk of prematurity or sga infants beyond that seen in general asthma population [ ] . while considering the possible anaphylaxis, this therapy is not initiated in pregnancy though sometimes may be continued if already in progress [ ] . in addition to the safety, there is still a notable problem, namely, the dose titration of omalizumab. omalizumab should be dosed according to body weight and pretreatment canadian respiratory journal serum total ige levels and is injected subcutaneously once every or weeks. each injection ranges from to mg, and the maximum dose is no more than mg every weeks in the united states of america and mg in the european union. in the european union, the eligible baseline ige level is - iu/ml. in all countries, body weight must be no more than kg. accordingly, patients whose body weight or baseline ige levels are not in the above range should not receive the treatment [ ] . for pregnant women with asthma, maternal weight changes persistently over a short period of time as the fetus grows; therefore, the method of adjusting the amount of omalizumab according to body weight is a challenge for maternal asthma. after the use of high-dose ics, approximately - % of uncontrolled asthma patients still have persistent airway eosinophilia. accumulating evidence suggests that interleukin-(il-) contributes to airway eosinophilic inflammation. in addition to omalizumab, another two anti-il- monoclonal antibodies, mepolizumab and reslizumab, have also been approved as maintenance treatment program for patients with uncontrolled, persistent eosinophilic asthma with an exacerbation phenotype. mepolizumab has been approved by fda for maintenance therapy of severe asthma patients aged or older and can reduce exacerbations by approximately % in patients with severe asthma [ ] . and reslizumab is also fda approved for maintenance therapy of severe asthma in patients aged or older [ ] . benralizumab, another humanized monoclonal antibody selective for il- receptor, has also been approved by fda for add-on maintenance treatment of severe asthma in patients aged or older with an eosinophilic phenotype. ere are no adequate studies of monoclonal antibodies such as mepolizumab, reslizumab, and benralizumab use in pregnant women due to the fact that monoclonal antibodies can cross the placenta during the third trimester. however, in a study of cynomolgus monkey, no harmful effects were observed on the fetus or on neonatal growth up to . months after birth when monkeys were given iv benralizumab every weeks which is the maximum recommended human dose throughout pregnancy [ ] . collectively, there is rarely report on the use of monoclonal antibodies against il- in asthma during pregnancy and their adverse effects on the maternal health or the development of the offspring. ait is currently considered to be the only treatment for the etiology of asthma, which relieves asthma symptoms and reduces asthma attacks by regular subcutaneous injection, oral administration, or sublingual administration of an injection or oral preparation of known allergens. for the general asthma patients whose symptoms are well controlled or partially controlled and are clearly linked to a relevant allergen [ ] , it is a valuable treatment challenge to apply ait after weighing the pros and cons in order to improve asthma symptoms, lung function, or bronchial hyperreactivity and reduce medication requirements, but for the pregnant women with asthma, we should be more cautious. a review reports that uterine cramps may occur during anaphylaxis, also there are possible side effects of venom immunotherapy, and a year-old woman under wasp venom desensitization has a premature birth in her th week of pregnancy [ ] . it is generally recognized that ait should not be initiated during pregnancy because the risk of anaphylaxis is unknown and the benefits appear minimal. however, it can be continued in patients who already received an allergic vaccine, on a stable and nonescalating dose, and whose symptoms appear to improve [ ] . a group of pregnancies from atopic mothers who have received immunotherapy during pregnancy are studied retrospectively. e incidence of prematurity, toxemia, abortion, neonatal death, and congenital malformation is not greater than that for the general population. it suggests that ait can be cautiously continued during pregnancy without significant risk to either mother or fetus [ ] . ere are still studies concluding that ait (sublingual or subcutaneous) is safe during pregnancy, even when initiated for the first time in a pregnant patient. a retrospective study included patients ( pregnancies) receiving subcutaneous immunotherapy and pregnant patients ( pregnancies) without receiving immunotherapy. in the treatment group, the incidence is similar to or less than the general population with regard to abortion, perinatal mortality, prematurity, toxemia, and congenital malformation. in the control group, there is a higher incidence of abortion, prematurity, and toxemia as compared with those treated with immunotherapy. in addition, seven patients of the treatment group who are already pregnant when immunotherapy is initiated did not develop any complications and all delivered normally [ ] . in a prospective study, the subjects are divided into three groups: the treatment group receiving slit (sublingual ait, n � ) with either house dust mite or a mixture of up to five allergens, of which pregnancies received sublingual immunotherapy for the first time during pregnancy, the control group a received budesonide μg twice daily (n � ), and the control group b receives rescue treatment with salbutamol (n � ). after follow-up for six years, the incidence of perinatal deaths, prematurity, and toxemia of pregnancy is less than that in the general population but the incidence of complications is higher in both control groups. none of the patients develops complications during pregnancy and none has reactions to slit [ ] . asthma attack in pregnancy is defined as requiring medical intervention during pregnancy, including unscheduled doctor visits, emergency department presentations, hospitalization, or requirement for ocss, with the occurrence rate of at least %- %, and %- % of women suffering severe attack (requiring ocs). e risk of severe attack in patients with mild, moderate, and severe asthma is %, %, and %, respectively, reported by a prospective cohort study of women with asthma and pregnancy. and attack during pregnancy occurs mainly in the late second trimester, a mean of . ± . weeks of gestation. e major triggers are viral infection and nonadherence to ics; besides, the undertreatment of asthma and changes in hormone levels also play a role [ ] [ ] [ ] . attack of asthma during pregnancy is connected with increased risk of adverse pregnancy and perinatal outcomes. current studies have shown that acute attack increases the risk of lbw; however, the relationship between acute attack and other adverse outcomes is still controversial, such as pb, sga, congenital malformations, neonatal death, and hospitalizations, as well as a range of maternal and placental complications, preeclampsia, gestational diabetes, placenta previa, and caesarean section [ , ] . for asthma management, early identification of asthma attack during pregnancy is very important. symptoms worsening is the main manifestation of the patient, combined with pef monitoring and attention to fetal activity which is helpful in judging the condition. if pef decreases more than %, personal predicted suggests severe exacerbation [ ] . patients with asthma in pregnancy can be treated by themselves in the early stage of asthma attack, and the specific steps and medication are the same as nonpregnancy asthma. once the patient's asthma symptoms are poor relieved or even worse, they should be hospitalized in time. in order to avoid hypoxia of pregnant women and fetuses, consider the following: giving oxygen inhalation to pregnant women, continuously monitoring oxygen saturation and fetal heart rate, using saba and ics actively, adding ocs if the patients are with poor effect, and even using intravenous hormone as soon as possible for the patients with respiratory failure. management. % of asthmatic pregnant women will not have an acute attack during childbirth, and only inhaling bronchodilators can control symptoms, while patients with chronic or frequent use of steroids may be at risk of adrenal insufficiency during delivery, and a pressure dose ( mg of hydrocortisone every - hours) can be used to prevent adrenal insufficiency during delivery and on the first day after delivery. furthermore, if mothers use saba in large doses, newborns, especially premature babies, need to be tested for blood glucose within hours. in addition, asthma patients should also be cautious about using some common measures during delivery such as drugs for cervical ripening, analgesic, and anesthesia. oxytocin and prostaglandin e can be to induce labor in asthmatics. fentanyl is an acceptable drug to relieve pain; however, morphine, meperidine, or other narcotics for analgesia are likely to cause the release of histamines and bronchospasm. epidural rather than general anesthesia is recommended for asthmatics who need a painless delivery to reduce the risk of pulmonary infection and atelectasis [ , ] . intervention is equal in importance with the pharmacologic treatment. as mentioned at the beginning of the article, poor drug compliance and incorrect use of inhaled drugs are important factors that make asthma difficult to control, especially in pregnant women, given the potential risks of drugs to mothers and fetuses. e purpose of education is to improve patients' compliance, standardize medication according to the asthma action plan, master correct inhalation techniques, and self-monitor the condition. triggers. asthma patients should avoid or reduce irritants such as allergens, climate change, drugs, sports, specific environments, and occupations and try specific environmental controls such as air filtration or special bed covers. in addition to the above factors, avoiding infection is an important measure to reduce asthma attacks. viral infection has been identified as an important trigger for asthma attacks, and the risk of viral infection in asthma during pregnancy is higher than that in nonpregnant women. a prospective cohort study of asthmatic pregnant women and nonasthmatic pregnant women finds that % of asthmatic pregnant women and % of nonasthmatic pregnant women have a common cold during pregnancy (incidence rate ratio: . , % ci: . - . , p < . ). among asthmatic pregnant women, rhinovirus is detected in . %, coronavirus in . %, influenza virus a or b in . %, and enterovirus in . %. one-third of patients who tested positive for respiratory virus pcr are associated with acute exacerbation of asthma, and one-third lost control of their asthma [ ] . asthma patients often have comorbidities (like allergic rhinitis, sinusitis, gastroesophageal reflux, obesity, chronic obstructive pulmonary disease, bronchiectasis, obstructive sleep apnea syndrome, depression and anxiety, etc.), especially those with severe and uncontrolled asthma. in pregnant women, the prevalence of gestational rhinitis, gerd, overweight or obesity (in europe), and depression is %, - %, . %- . %, and %, respectively [ ] [ ] [ ] [ ] . since these comorbidities may lead to worsening of symptoms and drug interactions, even increases the risk of perinatal adverse outcomes in pregnant women, active treatment should be given and followed. for pregnant women, physical structural changes, the direct effect of hormones, and the changes of immune function induced by hormones are involved in the control of asthma; one-third of asthma patients are aggravated due to pregnancy. to minimize maternal and fetal perinatal risks, assessing patient's condition by early identification of new onset asthma in pregnancy, monitoring changes of asthma symptoms, pef variability, feno, and fetal activity, education for improving patients' compliance and standardizing medication, avoiding triggers, treating complications actively, and using drug therapy by a stepwise approach are all needed to be written in asthma control plan. many studies and guidelines support the fact that inadequate treatment has a greater impact on mothers and fetuses than potential drug side effects. achieving a good whole-course management of asthma patients in pregnancy still requires continuous efforts of clinicians and patients themselves. canadian respiratory journal conflicts of interest e authors declare that they have no conflicts of interest. asthma prevalence among pregnant and childbearing-aged women in the united states: estimates from national health surveys effect of maternal asthma on birthweight and neonatal outcome in a british inner-city population e course of asthma during pregnancy in a recent, multicase-control study on respiratory health asthma knowledge, care, and outcome during pregnancy: the qakcop study asthma in pregnancy: a hit for two perinatal outcomes following maternal asthma and cigarette smoking during pregnancy pulmonary physiology in pregnancy alterations in physiology and 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an eaaci position paper arguing the misconceptions in allergen-specific immunotherapy e safety of immunotherapy during pregnancy a retrospective study on the safety of immunotherapy in pregnancy a prospective study on the safety of sublingual immunotherapy in pregnancy severe asthma exacerbations during pregnancy asthma exacerbations during pregnancy: incidence and association with adverse pregnancy outcomes asthma during pregnancy: exacerbations, management, and health outcomes for mother and infant determinants of low risk of asthma exacerbation during pregnancy asthma management across the life span: the childbearing woman with asthma a prospective study of respiratory viral infection in pregnant women with and without asthma are proton pump inhibitors safe during pregnancy and lactation? evidence to date risks associated with obesity in pregnancy, for the mother and baby: a systematic review of reviews depression during pregnancy key: cord- -crkcxpw authors: ganasegeran, kurubaran; ch’ng, alan swee hock; looi, irene title: covid- in malaysia: crucial measures in critical times date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: crkcxpw nan m alaysia contracted a high number of covid- positive cases among the southeast asian countries. albeit the global covid- pandemic trend is increasing, malaysia is seeing a decrease on the number of infections, with high recoveries and low mortality rates [ ] . this viewpoint aims to discuss the targeted containment strategies executed by malaysia, which till date is showing positive responses in controlling the spread of covid- . as of july , , malaysia recorded covid- positive cases, with deaths and recoveries, leaving with only active cases [ ] . the first wave of the outbreak (january , -february , ) reported cases and constituted mostly of imported cases [ ] . malaysia noted zero new cases until february , , however, beyond this date marked the beginning of the second wave that observed an exponential rise of daily positive cases. as of april , , malaysia recorded positive cases with deaths, being one of the highest across the southeast asian countries [ ] . figure shows time trends of positive cov-id- cases overtime, since the first day when malaysia contracted a positive case up to july , . the figure shows a declining trend with the implementation of the four phase (reviewed every two weeks apart in view of covid- incubation period of fourteen days) movement control order (mco) that was enacted since march , . with declining trends, malaysia further enforced the conditional movement control order (cmco) and the recovery movement control order (rmco) that have seen cases declining to single digits per day. however, periodic peaks were observed during the cmco phase due to detection of positive cases among migrants in detention centers (figure ) [ ] . measures to contain the outbreak were formulated based on three principal strategies: ( ) slowing introduction of global infections; ( ) slowing infection of local outbreaks; and ( ) executing community mitigation strategies. travel restrictions for citizens, suspension of immigration facilities to travelers from affected regions, isolation of confirmed cases and quarantine of exposed individuals were imposed to slow introduction of global infections [ , ] . entry and exit points were monitored closely with thermal scans installed to screen potential infectees [ ] . an exponential growth of covid- cases was triggered in the second wave (from february , ) due to a massive cluster gathering within the state of selangor [ ] . the movement control order (mco) that promulgated restriction of movement and activities under the prevention and control of infectious diseases act and the police act was enforced on march , [ ] . under mco, the following measures were implemented: ( ) prohibition of mass movements, religious, sports, social and cultural activities; ( ) closure of business premises except for daily necessities and needs services; ( ) selfquarantine and health check measures for those who returned from abroad; ( ) restrictions on tourists and visitors; ( ) closure of schools, kindergartens and higher institutions of learning; and ( ) closure of all government and private premises except for essential services like water, electricity, telecommunications, etc [ ] . spatial hotspots (red zones) with high incidence of positive infections (≥ cases) would trigger an enhanced movement control order (emco) (a full lockdown) [ ] . such measures slowed local infections and enabled health care authorities to perform contact tracing, identify potential clusters for investigation and optimize health care resources for screening and treatment. violators of the mco regulations are subjected to penalties under the penal code, which is under a federal gazette; where violators may receive a penalty of up to myr (approximately us$ at the time of writing) and/or up to six months imprisonment [ ] . with legislative and non-pharmacological interventions being put into place, the success of compliance was highly correlated with community engagement and support. the implemented interventions were concurrently executed with community mitigation strategies. key community mitigation measures include: ( ) obeying the cancellation or postponements of ad hoc or planned events, sports and religious activities; ( ) high compliance on the practice of physical distancing measures and the usage of face mask; ( ) reducing flight and public transportation services; ( ) self-quarantine at home; ( ) changes to crucial essential services like funerals to minimize crowd size and exposure to body fluids; and ( ) avoidance of misinformation -verified and clear information regarding covid- needs to be delivered on-time and consistently to the public to avoid fake news, rumors and panic. conveying accurate and rapid information on covid- risk factors, transmissibility, clinical features and prevention strategies via social, print or electronic media would enhance community's understanding of the disease. consistent daily interaction between the government and the people to digest latest statistical updates and the do's and don'ts during mco via short messaging service (sms) reminders, daily press conference and social media posts had maintained transparency and compliance for successful execution of legislative measures [ ] . the level of public commitment was appreciable when the malaysian government announced alternatives to the conventional bazaars during the fasting month with novel alternatives; such as to use food delivery services and e-services. this prevented massive gatherings that would increase the risk of infections. with declining trend of positive cases, malaysia executed a relaxed conditional movement control order (cmco) that aims to carefully re-open the country's major economy in phases [ ] . malaysia adopted the carefully crafted standard operating procedures (sops) based on the avoidance of cs (crowded places, confined spaces, close conversation) and the practice of ws (wash hands, wear masks, warn against risks, symptoms, prevention and treatment) during the enforcement of the four main legislation acts through the mco, emco, cmco and rmco till date, which have seen success in flattening the epidemic curve [ ] . contact tracing is more critical when individuals with high risk of transmission within detected clusters are difficult to track rapidly. other vulnerable groups such as migrants or homeless people with poor living conditions compounded with overcrowding have increased susceptibility towards the spread of potential local contagion [ ] . the execution of institutional quarantining of people who have been in contact with confirmed or probable cases may be unrealistic at certain times as this overwhelms the system and may lead to more infections. self-quarantine may be a more realistic measure [ ] . rigorous and routine screening efforts and disinfection process in public or overcrowded places is important to control the spread of the infection. as severe complications and high mortality rates is correlated with older people [ ] , more focused and targeted interventions of infection control efforts and potential treatment modalities should be prioritized to the aged population. specific and more comprehensive guidelines on hygienic processes and infection control strategies should be directed to elderly care homes. while there are guidelines in place [ ] , monitoring the compliance in these homes are crucial to prevent the spread of covid- among the elderly. lockdowns have unprecedented psychological repercussions to people [ ] . to overcome stress, anxiety, depression or isolation, malaysia has introduced psychological services through the establishment of care lines and virtual counseling sessions [ ] . misinformation cause panic, as such, it is crucial to provide verified information of the contagion through daily updates. we are in the midst of battling the transmission of a pathogen that disrespects national and international borders with many uncertainties. while being the most potent infection to the human population globally, the anticipated hazards and implications are expected to prolong for months to come until appropriate evidenced based treatment modalities and vaccines are discovered. pending answers to the many puzzles of covid- from the scientific and medical perspective, it is crucial to reckon the importance of executed public health measures in such critical times to disallow an uncontrolled spread and replication of the virus to the human living environment, which would ultimately be difficult to tackle. malaysia has taken a unique targeted approach in controlling the covid- outbreak. current situation of covid- pandemic in malaysia coronavirus disease (covid- ) pandemic covid- outbreak live updates coronavirus travel ban: malaysia stops visas for chinese travelers from affected areas. the star malaysia to announce additional measures to prevent spread of new china virus the prime minister's special message on covid- - press release: enhanced movement control order refugee and migrant health in the covid- response covid- and community mitigation strategies in a pandemic guidelines for care homes for older people in the context of covid- the psychological impact of quarantine and how to reduce it: rapid review of the evidence correspondence to: dr kurubaran ganasegeran clinical research center seberang jaya hospital ministry of health malaysia penang malaysia medkuru@yahoo acknowledgements: this article is dedicated with utmost appreciation to the government of malaysia, the min- the authors completed the icmje unified competing interest form (available upon request from the corresponding author), and declare no conflicts of interest. key: cord- -l pvf authors: collins, charles; xu, jing; tang, shenglan title: schistosomiasis control and the health system in p.r. china date: - - journal: infect dis poverty doi: . / - - - sha: doc_id: cord_uid: l pvf over the last sixty years advances have been made in the control of schistosomiasis in p.r. china. there are, however, difficult challenges still to be met. this paper looks at the extent to which the health system offers a positive environment for the control of the disease. it starts by tracing three phases in schistosomiasis control: disease elimination strategy through snail control ( s-early s); morbidity control strategy based on chemotherapy (mid s to ); integrated control strategy ( +). each one of these phases took place in distinct policy-making environments. the paper partly draws on these phases to set out five issues of disease control and discusses them in the context of the health system and its recent trends. these cover the policy-making process, intersectoral action for health, equity and access to health services, funding for public goods and externalities, and strengthening resource management and planning. these issues form the basis of an agenda for integrating research and capacity strengthening in the chinese health system with a view to creating a more positive enabling environment for schistosomiasis control. in so doing it is important to emphasize the role and integrity of the public sector against its commercialization, the underlying value of equity, a systems wide perspective, and the role of advocacy. schistosomiasis japonica is mainly prevalent in people's republic of china (p.r. china), the philippines and small pockets of indonesia, although p.r. china is the most heavily endemic of the three countries [ , ] . a large-scale epidemiological survey at the beginning of s found that the disease was endemic in provinces, one autonomous region and one municipality (city) mainly along the yangtze river in the south of china [ ] . it was estimated that . million people were infected with schistosomes and more than million people were at risk of infection in the s. there were . million infected cattle and the habitat area of oncomelania hupensis, the intermediate host snails of s. japonicum, reached . billion m . a great deal has been achieved in controlling schistosomiasis in p.r. china [ , , ] . between and disease transmission was interrupted in five provinces namely guangdong, shanghai, fujian, guangxi, and zhejiang. by , three provinces -sichuan, yunnan, and jiangsu -reached the criteria of transmission control (both human and livestock prevalence less than %). four other provinces characterized with complicated environments and easily affected by the water level of the yangtze river-hubei, hunan, jiangxi and anhui -reached the criteria of infection control in (both human and livestock prevalence less than %). the number of infected cases has been reduced by over % since the s, reaching the lowest historic level of , infections in [ ] . the area of oncomelania hupensis habitats was estimated to be . billion m which is about % of that in s [ ] . having noted the above achievements, there are still many major challenges such as the existing extensive snail habitats with complicated environments, ecosystem changes caused by the construction of the three gorges dams and the south-north water conversion project, the effects of climate change, the scarcity of a highly sensitive surveillance and response system, and the access of infected persons to health care. this paper analyses the extent to which one of the keys to understand these challenges rests not only in the confines of the schistosomiasis diseases control programme, but in the rest of the health system. how diseases control programmes fit into health systems has been a recurrent theme of health systems analysis for many years. the debate over the vertical and/or horizontal nature of disease control programmes has occupied an important place in health systems analysis, together with discussions over the nature of integration and the specific circumstances in which integration is or is not appropriate [ ] [ ] [ ] [ ] . recent work has also looked to develop a more synergistic interrelationship between disease control programmes and the rest of the health system in addition to a more systems approach [ ] [ ] [ ] [ ] . this paper comes within this line of analysis and focuses on a particular aspect; namely the extent to which the health system as a whole provides a positive environment for the effective development of disease control. it is based on a review of existing research, the analysis of which takes into account the research and practical experience of the authors. following this introduction, the paper identifies the historical phases in the control of schistosomiasis in p.r. china. the phases operated in specific political and social contexts and exhibited particular approaches to disease control. this leads to a consideration of an 'enabling environment' that we judge to significantly further disease control. on this basis, our analysis explores the extent to which the health system does or does not meet the needs of this 'enabling environment'. attention is paid to the policy-making process, intersectoral action for health, equity and access to health services, funding for public goods and services, and strengthening resource management and planning. where appropriate, recommendations for an agenda of health systems research and development will be made. the paper concludes by analysing four emerging themes; the role and integrity of the public sector, the importance of equity for infectious diseases of poverty, the significance of health systems development, and the importance of advocacy. the paper is aimed at researchers, policy-makers and practitioners concerned with both schistosomiasis control and health systems development. it also suggests a line of analysis that can be developed in the analysis of other infectious diseases and their control, such as tb, malaria and hiv/aids. schistosomiasis was one of the serious infectious diseases at the time of the founding of the p.r. china in . many famous terms, such as "village without villagers" "widows villages", and "big-belly village", were used to describe the devastating consequences the disease brought to the chinese people, especially the poor [ ] . since the s china has been fighting the disease; strategies and approaches have evolved in a context of political, socio-economic, technological and epidemiological change. three relatively distinct phases may be identified in the process of disease control: a) s to early s, b) mid- s to about , c) from onwards. in identifying these phases we recognise a degree of generalisation in the analysis together with the overlapping nature of the phases. each phase not only possesses an emphasis on certain approaches to disease control but relates to a policy environment of political and social change. disease elimination strategy with an emphasis on snails control ( s to early s) confronted by the poor state of health in p.r. china and with schistosomiasis as one of the main infectious diseases, there was strong political will among the leaders of the new republic to control the disease. however, the financial and human resources for healthcare in p.r. china were very limited, and most of them were distributed in a few urban cities. the infrastructure of china's health systems in most places was poor and not up to the level of providing appropriate healthcare to the vast majority of the population. however, cooperation among different sectors was developed with programs receiving political support from a high level. in such circumstances, the ministry of health developed a policy of "prevention first" in the s and focused on snail control. snail elimination with environmental modification and mollusciciding was emphasized in combination with chemotherapy. mass movements were developed to mobilize community resources to contribute to the snail control campaign, through free labour, and local-driven innovative models for snail elimination. under the auspices of the communist party, the chinese people, as well as paramedics, such as barefoot doctors at the village level, engaged in disease control programmes. in the meantime, agricultural engineering and water conservancy activities, such as reclaiming wetlands, digging new ditches and filling the old ones, and changing rice paddies into dry crops were developed and implemented as a series of concerted actions to modify the snails' habitats to be unsuitable for living and breeding. an important feature of this disease control was the establishment and the operation of the vertical national schistosomiasis control programme in the s. from national to provincial, prefectoral, county and township levels, anti-schistosomiasis institutions or stations were set up to take the main responsibility for disease control and treatment. the number of staff specialized in prevention and clinical care and working in these specialized organizations reached , by the mid s, a powerful workforce fighting against the disease. the national programme, including these institutes and stations, was relatively well funded until the late s. as results of the effective interventions, a large number of places in p.r. china were free of snails. the prevalence rate of schistosomiasis and new cases was reduced to a very low level in the early s, particularly in the east coastal areas of china. china launched its economic reform in , transforming its planned economy into a market one. the collective economy, based on the commune system in the rural areas, collapsed. a de facto privatization of agricultural production, named as "household responsibility system" was introduced in - in almost all the townships and villages. chinese society has undergone profound changes since the economic reform. while many of these may be viewed as positive, such as improved living standards, there are important downsides, such as worsening equity and social justice. the diminished collective economy in the rural areas meant that the community-based health insurance schemes, called "cooperative medical scheme", collapsed in over % of townships and counties by the middle of the s. government health facilities received relatively less funding to cover their operational costs, while they were implicitly encouraged to increase service charges to support the provision of health services. the commercialization of healthcare has become widespread and common practice in health facilities in p.r. china while the health policy of "prevention first" has to a large extent been neglected since the economic reform. service providers became interested in generating revenues through service charges and profits from drug sale to cover costs and increase their incomes that were often linked to the level of revenue generation. although the government still gave some support for control of schistosomiasis, but it was limited and could not meet the needs for disease control. even the anti-schistosomiasis institutions and centres were required to generate revenues to cover partial costs of their operations. few health facilities in p.r. china were still interested in engaging in preventive measures to control schistosomiasis, among other diseases. furthermore, the mobilization of community resources for the disease control was no longer easy. free labour was no longer available to tackle snail problems in rural communities, as the township and village leadership no longer had powers to force farmers to work on community projects for free. in addition, intersectoral action for health (iah) became difficult, if not impossible. market mechanisms have now come to dominate production in chinese society, while the political push to promote intersectoral action for health and social development, which was strong in the planned economy, has been greatly weakened. by the late s, some provinces and counties had scaled down the vertical programme and integrated many anti-schistosomiasis stations into general centre for disease control (cdc) systems or other disease prevention institutes. by co-incidence, the who expert consultation committee for schistosomiasis control in adjusted its strategy and objectives for schistosomiasis control from transmission interruption or elimination to morbidity control in developing countries [ , ] . the new strategy focused on changing people's behaviour with an objective to reduce the morbidity and mortality of schistosomiasis, rather than controlling the transmission of schistosomiasis completely; it was convinced that it would be formidably difficult to eliminate or interrupt the transmission of schistosomiasis without an enormous amount of financial investment in developing countries. with the support from the world bank loan ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , the implementation of the strategy, emphasizing chemotherapy treatment of human being and livestock, as the main approach, was initiated in and completed in . different chemotherapy strategies were carried out in different endemic areas: mass chemotherapy was used for the people from endemic areas with a high prevalence and with a history of water contact. for residents and bovine in areas with medium endemicity, the treatment was only given to those with stool egg positive or positives in serological tests. in the area of low endemicity, only children were screened and treated, if diagnosed as positive cases. infected cattle were also given appropriate treatments under the world bank project [ ] . the achievement of the world bank funded project proved that the chemotherapy based strategy could decrease the prevalence of schistosomiasis quickly but that the consolidation task is arduous, as the areas of snail habitats are still large and fluctuated greatly in p.r. china [ ] [ ] [ ] . potential transmission remains considerable in the lake areas. in addition, the drugs for anti-schistosomiasis was free under the world bank project, other costs of healthcare such as drugs for liver protection, were required to pay out of pocket by the patients. in the context of lack of appropriate health insurance schemes put in place, early case detection was often problematic. in a more general sense, access to healthcare in p.r. china was worsening in the s, as seen in a rapid rise of healthcare costs, and increasing financial challenges the health insurance schemes in both urban and rural areas were facing. following the completion of the world bank project, the central government failed to come up with concrete policies supporting sustainable control of schistosomiasis, which leading to the resurgence of the schistosomiasis transmission after the world bank loan project ended [ ] [ ] [ ] . since the late s, the government of p.r. china has increasingly recognized the important role the state should play in developing and strengthening health systems to improve equitable access to healthcare for the vast majority of the population. this responded to the increased inequity in health and healthcare and the resulting discontent among the public. the outbreak of sars in was another alarm signal to the government that public health crises can affect not only health but also economic growth. therefore, strengthening health systems to achieve universal health coverage has been put on the political agenda. a decision on the reestablishment of rural health insurance schemes with financial support from central government in was one of many examples that the government of p.r. china has once again taken health policies seriously. under these circumstances and with the re-emergence of schistosomiasis at the beginning of st century in p.r. china, schistosomiasis control has once again been given a high priority. it was recognised that the chemotherapybased approach could reduce the prevalence/morbidity to a low level rapidly. however, the environment of snail habitats would not change much and the opportunities of reinfection for many at-risk population remained high due to unchanged agricultural production and people's life styles in endemic areas. hence, a new integrated control strategy aimed to interrupt transmission based on reducing the rate of transmission of schistosomiasis infection from cattle and human being to snails has been developed and adopted by the national schistosomiasis control programme. the interventions include agricultural mechanization (to replace the use of cattle), supplying water, sanitation and lavatories/ latrines, providing boats with faecal-matter containers, plus routine chemotherapy, moulluscides and health education [ , ] . these interventions have been made possible, owing to strong political, policy and financial support given to the national schistosomiasis control program in recent years. this forms part of the new health system reform in which increasing equitable access to public health interventions is one of top priorities set out by the government. after several years implementation of the integrated control strategy, positive achievements have been seen. four more provinces have now reached the level of infection control and three have met the targets of transmission control [ ] . compared to the situation in , the number of estimated infected people reduced from , to , . the number of acute cases decreased from , in to only in [ ] . the prevalence rate of infected cattle reduced from . % to % over the period. such results show that political will and appropriate policy on effective strategy on schistosomiasis control are critically important to effective disease control. equally important is the strengthening of overall health systems from national to local level. marketertization of healthcare, particularly public health programmes, would not work, especially in infectious disease control. this section will partly build on section by drawing out and discussing five key issues of schistosomiasis control in p.r. china and how they relate to the health system. it will take into account the current trends in the health system and ask how these affect schistosomiasis control. it will also provide the grounds for recommending key areas of research and capacity development in the health system and in relation to schistosomiasis control. although a vertical form of schistosomiasis disease control programme was set in phase one (see section ), there has been a process of integrating the activities of disease control with the cdc system and the general health services. a first area of research and policy analysis therefore is to analyse the extent of that integration, mapping out how this is expressed in resource generation and allocation, policy-making and planning, resource management, service delivery, and governance. this may be accompanied by an analysis of the determinants and impact of the diverse forms of integration in operation [ ] and how the analysis of the five factors in this section would affect the degree of integration. section raised important issues about policy-making for and the prioritising of schistosomiasis control in p.r. china. it was noted that different strategies of intervention have been used corresponding with the three phases which, in turn, corresponded with different policymaking environments. although the first phase was characterised by depleted national resources, there was strong political will to control the disease; a robust vertical disease control programme was developed along with effective community involvement in transmission control, and intersectoral action for health. the second phase occurred in the context of market reform characterised by health care commercialisation and the loss of intersectoral action for health and community involvement. the phase coincided with a who supported shift to morbidity control through the world bank supported project. the third phase constitutes a reaction to public health crises and the problems of inequity engendered through market reform and health care commercialisation. the priority given to schistosomiasis control and the sustainability of interventions are certainly critical issues. these are important given the severity of the disease but also because it can easily rebound if attention is reduced. section emphasised the differences between the high priority given in the first and third periods and the lesser priority given in the second period. it goes without saying that policy-making operates in an historical context and the three phases clearly confirm this. yet this raises a challenge to policy-making; how to achieve consistency and sustainability to undertake the medium to long term preventive interventions and intersectoral action for health as a form of disease control that goes beyond the shorter term political changes and the periodic form of loan financing. at the same time, the possibility of the disease to rebound requires a more sophisticated process of priority setting that relies primarily on the more immediate indicators of mortality and morbidity. prioritising schistosomiasis control within the broad range of health needs and interventions has to go beyond the spoken and written word of the policy declaration. resource allocation has to give a material backing to the prioritising. neither can this material backing be based on the assumption that regions and localities are able to raise their own funds for diseases control. this is particularly true for areas of central and western china which already receive central subsidies for rural health insurance, among others. schistosomiasis is more prevalent in the poorer regions of china; ross et al. noted that the disease ". . .remains a major problem in the marshland and lake areas of hubei, hunan, anhui, and jiangxi and in some mountainous areas of sichuan and yunnan" [ ] . these are precisely among the regions of china that particularly require such subsidies and central funding. lastly, we need to emphasise that schistosomiasis control requires joint policy-making work across organisational boundaries and systems. policy-making needs to move beyond the confines of the health system, a point we now pick up under the heading of intersectoral action for health. iah is clearly important and comes through strongly as an important requirement of the health system for schistosomiasis control. the headlong imperative of economic growth can lead to growing inequality, poverty and disease burden. the precipitous implementation of water development projects and increased urbanisation has laid the conditions for increasing mortality and morbidity from schistosomiasis in p.r. china. to undercut the social and economic conditions of the disease requires a broad political perspective that puts the disease on the policy agenda, brings the disease control into the broader policy process identifying its social and economic conditions and secures the links between the disease control and health systems development. this was clearly more evident in the first and the third phases outlined in section . in the push for increased mechanisation in agriculture and improved water and sanitation, the health system needs to play its part in breaking the barriers within the health system and between the health and other systems such as agriculture, forestry and water / sanitation. it requires advocacy by actors involved with schistosomiasis control through the generation and presentation of evidence on the social economic conditions of schistosomiasis, policy analysis on the effectiveness and feasibility of interventions, together with networking and building coalitions of support for the control of the disease [ ] . there is a need for this advocacy to be based on the underlying values of effective health interventions through iah and equity. an important constraint on iah in p.r. china is the inward looking commercial practices of government sectors since the economic reform. concerned with revenue growth and surplus generation, little is left for pooling resources and space for working together. while recent years have seen improvements in the central coordination of government actionsthrough the national leading group for schistosomiasis control and the five year plan for schistosomiasis controlthere is a need to monitor and evaluate the effectiveness of these organisational changes in leading to implementation of iah at central, regional and local levels. schistosomiasis is principally a disease of the poor and china is far from being an exception in this respect [ , [ ] [ ] [ ] . this raises a number of important issues: the extent to which there is an overall focus in the health system (and within schistosomiasis control) on equity and the poor, the social determinants of schistosomiasis and the impact the disease has on poverty, and the access to treatment for the poor. those infected may use the schistosomiasis control stations (scs) although many of these have been integrated into the centres of disease control (cdc). treatment for schistosomiasis in these facilities is free at the point of delivery. however, before reaching these facilities, patients often pass through general health facilities (e.g. village health stations). there are certainly financial restrictions to access to these general health facilities for the poor, which can lead to further transmission of the disease mainly among the poor. the problem is that yu et al's study in villages of hunan province in found ". . .both the willingness and the amount that people were willing to pay for treatment were low among villagers in the endemic areas in this region, especially in heavily endemic areas where villagers are most affected and have the lowest ability to pay" [ ] . although many are covered by the diverse forms of government sponsored health insurance in p.r. china, regulations on co-payments and ceilings negatively affect access. linked to this are cases of rent seeking behaviour by providers such as supplier induced demand and mark-ups on medicines. research into treatment of tb patients has also shown a certain lack of interest by general health care providers in referring patients on to the free care at the scss and cdcs, thus helping their own health care facility to reap the financial rewards [ ] . whether this occurs in the case of schistosomiasis needs to be the subject of research. the control of schistosomiasis requires iah, interventions that provide public goods and services together with goods with high externalities. this suggests the importance of appropriate and secure funding for disease control by the state and based on taxes or similar secure revenues [ ] . these forms of disease control are not appropriate to financing and provision through private markets or commercialised public provision. the commercialisation of the health system in recent decades raises critical issues for schistosomiasis control in p.r. china. restrictions and relative decreases in government budget allocations to health facilities, the increased dependence on user fees and insurance payments, the use of staff bonus schemes in health facilities based on treatment, the move from prevention to more revenue earning curative services, and the emergence of supplier induced demand raise serious doubts over the compatibility of schistosomiasis control and commercialisation of the public sector in p.r. china [ ] . in theory, cdc and similar preventive institutions in p.r. china should be fully funded by the government. in reality, however, a vast majority of cdc need to raise some fund through service charges in order to cover partial operational costs and increase bonus payments to their staff. a key issue here is the need to develop general funding mechanisms based on tax bases and centrally or regionally allocated to affected regions to allow for the provision of public goods and preventive interventions required in the control of schistosomiasis. complementing the four previous factors of health systems development, there are a number of key areas in which the management and planning of resources need strengthening. these include surveillance and monitoring, human resource development for both specialist research and development in schistosomiasis control and the training of general health staff in the disease control, and the supply systems for delivering medicines. monitoring the effectiveness of implementing schistosomiasis control is a key challenge. there are two important areas for development: firstly, there is the need to ensure targets met with the delivery of high quality services, and secondly, to ensure efficiency in resource utilisation. another most important issue is how to improve positive synergies of combining the local resources with the fund from the central government. some interventions, such as mollusciciding, chemotherapy of local residents and bovines, and faecal management need appropriate resource pooling to increase the population coverage, while some engineering related interventions, such as agriculture irrigation system modification, altering the crops planting, biogas station should pool resources from different channels in an effective way. the high cost-effectiveness for a specific strategy in the either vertical or integrated control program relies on good resource management. in the current status of p.r. china, it is essential for the national control programme in p.r. china to be part of the push for universal coverage of health care in order to ensure sustainable control of schistosomiasis in p.r. china. the five issues featured in the previous section constitute an agenda for integrated research and capacity strengthening in health systems with a view to schistosomiasis control. applied research should increase our understanding of the health system needs of schistosomiasis control, while development strengthens the capacity of the health system to meet the needs of disease control. in so doing, there are four important considerations. a) an important theme running through this paper is to shift away from the commercialisation of the public sector and move towards strengthening of the role and integrity of the public sector in schistosomiasis disease control. this is apparent in the strengthening of policy-making in government, intersectoral action for health, the importance of equity as a key value, the access to treatment, the provision of public goods and the strengthening of resource management and planning. these need to be broken down into specific measures, such as strengthening surveillance systems, urban and rural health insurance, and funding for public goods and services. b) an underlying value of the whole approach to the control of schistosomiasis is that of equity. on the one hand, this requires a reaffirmation of public service values around health systems based on improved and more equitable health and health care. on the other, it is a disease of poverty, the control of which needs to get to the foundations of that poverty. c) the call for research and development in these five areas should not lead to isolation of specific forms of disease control. many of these features of disease control hold for other diseases. at the same time, care needs to be taken in dealing with these issues in a system wide perspective. for example, setting the priority of schistosomiasis control in policy-making and resource control needs to be seen in the context of general health needs and general health planning. the exercise of health needs identification needs to be done across the full spectrum of infectious disease of poverty and, in fact, the overall health needs of a society. the exercise conducted in this paper needs to be one of many exercises in disease control and in which health systems change is responding to health needs of a population. d) lastly, we return to advocacy. it is anticipated that the discussion of this and other likeminded papers should lead to 'a health systems agenda for schistosomiasis control'. such an agenda needs to coalesce around a coalition of stakeholders; those support of sufferers of the disease, researchers from diverse disciplines, technical experts, politicians, health managers and planners, and service providers. additional file : multilingual abstracts in the six official working languages of the united nations. the global status of schistosomiasis and its control kestens l: human schistosomiasis schistosomiasis control in china the global epidemiological situation of schistosomiasis and new approaches to control and research schistosomiasis control: experiences and lessons from china schistosomiasis endemic status in people's republic of china in a brief introduction to a nation-wide sampling survey on schistosomiasis vertical vs horizontal health programmes in africa: idealism, pragmatism, resources and efficiency delivery of priority health services: searching for synergies within the vertical and versus horizontal debate a code of best practice for disease control programmes to avoid damaging health care services in developing countries the 'diagonal' approach to global fund financing: a cure for the broader malaise of health systems? in maximising positive synergies between health systems and global health initiatives. edited by who. geneva: who; . . who: report on the expert consultation on positive synergies between health systems and global health initiatives. in maximising positive synergies between health systems and global health initiatives disease control and health systems in low-and middle-income countries: enhancing positive interrelation who: systems thinking for health systems strengthening who: world health organization new strategy on schistosomiasis control who: the control of schistosomiasis schistosomiasis control in china: the impact of a -year world bank loan project study on the re-emerging situation of schistosomiasis epidemics in areas already under control and interruption challenges and control strategies in areas of schistosomiasis transmission controlled and interrupted in china the public health significance and control of schistosomiasis in china-then and now epidemiology of schistosomiasis in the people's republic of china schistosomiasis japonica control and research needs re-emerging schistosomiasis in hilly and mountainous areas of sichuan china's new strategy to block schistosoma japonicum transmission: experiences and impact beyond schistosomiasis a strategy to control transmission of schistosoma japonicum in china integration of targeted health interventions into health systems: a conceptual framework for analysis. health policy plan schistosomiasis in the people's republic of china: prospects and challenges for the st century valuing health systems: a framework for low and middle income countries social science implications for control of helminth infections in southeast asia spatial epidemiology in zoonotic parasitic diseases: insights gained at the st international symposium on geospatial health in lijiang prioritizing research for "one health-one world is equity being sacrificed? willingness and ability to pay for schistosomiasis control in china how affordable are tuberculosis diagnosis and treatment in rural china? an analysis from community and tuberculosis patient perspectives market reform: a challenge to public health-the case of schistosomiasis control in china schistosomiasis control and the health system in p.r. china. infectious diseases of poverty we would like to express our sincere appreciation for constructive comments and suggestions on earlier version of the manuscript provided by prof. zhou xiao-nong. abbreviations cdc: centre for disease control; iah: intersectoral action for health; sars: severe acute respiratory syndrome; scs: schistosomiasis control stations; tb: tuberculosis. the authors have no competing interests.authors' contributions cc, the honorary reader at liverpool school of tropical medicine, participated in the initial design of the paper and in its writing, jx did the literature review used to develop the paper and also participated in the writing, and st participated in the initial design of the paper with cc, and drafted some sections of the paper. all authors reviewed and approved the final version. key: cord- - xrdv m authors: nowland, megan h.; brammer, david w.; garcia, alexis; rush, howard g. title: biology and diseases of rabbits date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: xrdv m beginning in , an inbred rabbit colony was developed at the phipps institute for the study, treatment and prevention of tuberculosis at the university of pennsylvania. this colony was used to study natural resistance to infection with tuberculosis (robertson et al., ). other inbred colonies or well-defined breeding colonies were also developed at the university of illinois college of medicine center for genetics, the laboratories of the international health division of the rockefeller foundation, the university of utrecht in the netherlands, and jackson laboratories. these colonies were moved or closed in the years to follow. since , the u.s. department of agriculture has reported the total number of certain species of animals used by registered research facilities ( ). in , , rabbits were used in research. there has been an overall decrease in numbers of rabbits used. this decreasing trend started in the mid- s. in , , rabbits were used in research. despite the overall drop in the number used in research, the rabbit is still a valuable model and tool for many disciplines. the rabbit has been utilized in immunology research for many years especially in regard to the structure of immunoglobulins and the genetic control of their formation. in addition, the rabbit is commonly used for the production of polyclonal antibodies for use as immunologic reagents (mage, ; pinheiro et al., ) . the relatively large body size and blood volume, easy access to the vascular system, and an existent large body of information on the purification of rabbit immunoglobulins are a few reasons the rabbit is preferred over other common laboratory animal species for polyclonal antibody production (stills, ) . the organization of the lymphoid system of the rabbit is comparable to that of other mammals. however, the rabbit does possess two gut-associated lymphoid tissues (galt) with specialized functions in the maturation of igm + b cells. these are the vermiform appendix at the distal end of the cecum and the sacculus rotundus at the ileocecal junction (mage, ) . for many years, the lack of rabbit-specific immunological reagents has limited the study of inflammation and immunity in the rabbit. the use of real-time polymerase chain reaction (rt-pcr) techniques has overcome this limitation and permitted such studies in many species other than man and mice. a quantitative real-time rt-pcr assay for measuring mrna for rabbit cytokines ifn-γ, il- , il- , il- , and tnf-α has been described (godornes et al., ) . recently, schnupf and sansonetti ( ) reported on rt-pcr primer pairs for analysis of three chemokines and ccl ) and cytokines (il- β, il- , il- , il- , il- , il- p , il p , il- a, il- f, . the profile of cytokines in the rabbit appears similar to other mammals. in mice and humans, the primary antibody repertoire is created by combinatorial rearrangement of a large number of immunoglobulin gene segments. other species (chicken, sheep, cattle, and rabbit) that have a limited number of gene segments utilize somatic gene conversion and/or somatic hypermutation (pinheiro et al., ) . in the former, a portion of the immunoglobulin gene is replaced with a gene sequence from a nonfunctional pseudogene. in the latter, single-nucleotide changes are made in the immunoglobulin genes (jenne et al., ) . in the rabbit, gene diversification occurs initially in the fetus and the neonate in sites such as the bone marrow. subsequently, between and weeks of age, immature igm + b cells undergo further diversification in the galt (appendix, sacculus rotundus, and peyer's patches) (mage et al., ; pinheiro et al., ) . furthermore, certain species of intestinal bacteria (bacteroides fragilis and bacillus subtilis) are required for appendix follicle development and antibody diversification to occur (hanson and lanning, ; mage et al., ) . most mammals express five classes of immunoglobulins: igm, igd, igg, iga, and ige. however, the rabbit lacks igd (sun et al., ) . the area of cardiovascular research has used the rabbit in a variety of different models. numerous dietary modifications will induce or exacerbate cholesterolinduced atherosclerosis in the rabbit. a brief overview of some of these dietary modifications can be found elsewhere (jayo et al., ) . research efforts into cholesterol metabolism have used the watanabe heritable hyperlipidemic (whhl) (atkinson et al., ; kita et al., ) and the st. thomas hospital strain rabbits (laville et al., ) . the whhl rabbit has a marked deficiency of low-density lipoprotein (ldl) receptors in the liver and other tissues. selective breeding of the whhl rabbit will increase the incidence of coronary artery atherosclerosis without increasing the incidence of aortic atherosclerosis (watanabe et al., ) . in contrast, the st. thomas hospital strain has a normal functioning ldl receptor but still maintains a hypercholesterolemic state (laville et al., ) . genetically modified rabbits have been created via both intracytoplasmic injection (li et al., ) and retroviral vectors (hiripi et al., ) . this has resulted in a multitude of new strains to address interesting research questions. cardiovascular disease (lombardi et al., ; peng, ; sanbe et al., ; stanley et al., ) including models of long qt interval for exploration of treatments (biermann et al., ; jindal et al., ; liu et al., a; peng, ; sanbe et al., ; ziv et al., ) and atherosclerosis (araki et al., ; masson et al., ; tjwa et al., ) are the main focus of model development. strains have also been developed that express human recombinant proteins in rabbit milk (chrenek et al., ; dragin et al., ; hiripi et al., ; houser et al., ; lipinski et al., ; simon et al., ; soler et al., ) . this ability can be passed down for multiple generations (chrenek et al., ; dragin et al., ) . these human proteins have resulted in antigen production for rotavirus vaccine creation, human factor viii that could be used to treat hemophilia (chrenek et al., ; krylov et al., ; simon et al., ) and human growth hormone that could supplement a deficiency in that hormone (lipinski et al., ) . rabbits that express enhanced green fluorescent protein (egfp) in various tissues have been created for the purpose tracking cells, which is important for tissue engineering and regenerative medicine studies takahashi et al., ; yin et al., ) . the mouth of the rabbit is relatively small, and the oral cavity and pharynx are long and narrow. the dental formula is i / , c / , pm / , m - / × = or teeth. a small pair of incisors is present directly caudal to the primary maxillary incisors and is referred to as 'peg' teeth. the peg teeth are used along with the primary incisors to bite and shear food. the absence of second incisors has been noted in some rabbit colonies as a dominant trait (i /i or i /i ). the teeth of rabbits erupt continuously throughout life and therefore will continue to grow unless normal occlusion and use are sufficient to wear teeth to a normal length. molars do not have roots and are characterized by deep enamel folds. rabbits normally masticate with a chewing motion that facilitates grinding of food by movement of the premolars and molars from side to side and front to back. the rabbit has four pairs of salivary glands, including the parotid, submaxillary, sublingual, and zygomatic. the parotid is the largest and lies laterally just below the base of the ear. the zygomatic salivary gland does not have a counterpart in humans. the esophagus of the rabbit has three layers of striated muscle that extend the length of the esophagus down to, and including, the cardia of the stomach. this is in contrast to humans and many other species, which have separate portions of striated and smooth muscle along the length of the esophagus. there are no mucous glands in the esophagus of the rabbit. although the stomach of the rabbit holds approximately % of the volume of the gastrointestinal tract, it is never entirely empty in the healthy rabbit. the gastric contents often include a large amount of hair ingested as the result of normal grooming activity. the stomach is divided into the cardia, fundus, and pylorus. the liver has four lobes. the gallbladder is located on the right. from the liver, the common bile duct empties into the duodenum posterior to the pylorus. rabbits produce relatively large amounts of bile compared to other common species. the pancreas is diffuse within the mesentery of the small intestine and enters the duodenum - mm distal to the common bile duct. the small intestine of the rabbit is short relative to that of other species and comprises approximately % of the total length of the gastrointestinal (gi) tract. because the gi tract of the rabbit is relatively impermeable to large molecules, kits receive most of their passive immunity via the yolk sac prior to birth rather than by colostrum. peyer's patches are found along the ileum, particularly near the cecal junction. the sacculus rotundus is a large bulb of lymphoid tissue located at this junction. the large intestine includes the cecum, the ascending colon, the transverse colon, and the descending colon. the ileocecal valve regulates flow of chyme into the cecum and retards reverse flow back into the ileum. the cecum is very large with a capacity approximately -times that of the stomach. the cecum ends in a blind sac, the appendix. the colon is divided into proximal and distal portions by the fusus coli, which serves to regulate the elimination of hard versus soft fecal pellets. hard pellets comprise about two-thirds of the fecal output. soft pellets, or 'cecotrophs,' have a high moisture content and are rich in nitrogen-containing compounds (ferrando et al., ) and the Β vitamins niacin, riboflavin, pantothenate, and cyanocobalamin. rabbits consume cecotrophs directly from the anus to obtain significant nutritional benefit. soft pellets are sometimes termed 'night feces,' since they are generally produced at night in domestic rabbits. in contrast, the circadian rhythm of cecotrophy is reversed in wild rabbits, occurring during the day when the animals are in their burrows (hornicke, ) . nostrils of rabbits are well equipped with touch cells, and they have a well-developed sense of smell. nasal breathing in rabbits is characterized by twitching of the nostrils at rates varying from to times per minute, although twitching may be absent in the relaxed rabbit. it has been speculated that inspiration occurs as the nostril moves up and that this serves to direct the flow of air over the turbinate bones where the olfactory cells are most concentrated. the musculature of the thoracic wall contributes little to respiratory efforts. instead, rabbits rely mostly on the activity of the diaphragm. because of this, artificial respiration is easily performed by alternating the head of the rabbit between the up and down positions, - times per minute, while holding the animal. compression and release of the chest wall is an ineffective means of artificial respiration in the rabbit. the pharynx of the rabbit is long and narrow, and the tongue is relatively large. these features make endotracheal intubation difficult. the procedure is further laboratory animal medicine complicated by the propensity of the rabbit to laryngospasm during attempts to intubate the trachea. the rabbit lungs consist of six lobes. both right and left sides have cranial, middle, and caudal lobes, with the right caudal being further subdivided into lateral and medial portions. flow volume of air to the left lung is higher than that to the right due to the lower resistance of the proximal airways per unit volume (yokoyama, ) . in rabbits, lung volume increases with age, in contrast to that of humans and dogs, in which it decreases. bronchial-associated lymphoid tissue (balt) is present as distinct tissue. a unique feature of the cardiovascular system of the rabbit is that the tricuspid valve of the heart has only two cusps, rather than three as in many other mammals. a small group of pacemaker cells generate the impulse of the sinoatrial (sa) node in the rabbit, a feature that facilitates precise determination of the location of the pacemaker (bleeker et al., ; hoffman, ; west, ) . the sa and atrioventricular (av) nodes are slender and elongated, and the av node is separated from the annulus fibrosus by a layer of fat (truex and smythe, ) . additional unique anatomic features of the cardiovascular system of the rabbit have been utilized to advantage. the aortic nerve subserves no known chemoreceptors (kardon et al., ; stinnett and sepe, ) and responds to baroreceptors only. because the aortic nerve, which becomes the depressor nerve, runs alongside but separate from the vagosympathetic trunk, it lends itself readily to implantation of electrodes (karemaker et al., ) . the blood supply to the brain is restricted mainly to the internal carotid artery. blood supplied via the vertebral arteries is limited. the aorta of the rabbit demonstrates rhythmic contractions that arise from neurogenic stimulation in a pattern related to the pulse wave (mangel et al., ) . the kidneys of the rabbit are unipapillate in contrast to those of most other mammals, which are multipapillate. this feature increases the ease with which cannulization is performed. the right kidney lies more cranial than the left. glomeruli increase in number after birth in rabbits, whereas all of the glomeruli are present at birth in humans (smith, ) . ectopic glomeruli are normal in the rabbit (steinhausen et al., ) . blood vessels that perfuse the medulla remain open during many conditions under which vasoconstriction of the cortical tissue occurs; thus, the medullary tissue may be perfused, while the cortex is ischemic (trueta et al., ) . the urine of adult rabbits is typically cloudy due to a relatively high concentration of ammonium magnesium phosphate and calcium carbonate monohydrate precipitates (flatt and carpenter, ) . the urine may also take on hues ranging from yellow or reddish to brown. in contrast, the urine of young rabbits is typically clear, although healthy young rabbits may have albuminuria. the urine is normally yellow but can also take on reddish or brown hues once animals begin to eat green feed and cereal grains. normal rabbits have few cells, bacteria, or casts in their urine. the ph of the urine is typically alkaline at about . (williams, ) . a normal adult rabbit produces approximately - ml/kg of urine daily (gillett, ) , with does urinating more copiously than bucks. the urethral orifice of the buck is rounded, whereas that of the doe is slit-like. this feature is useful for distinguishing the sexes. the testes of the adult male usually lie within the scrotum; however, the inguinal canals that connect the abdominal cavity to the inguinal pouches do not close in the rabbit. for this reason, the testes can easily pass between the scrotum and the abdominal cavity. this feature necessitates closure of the superficial inguinal ring following orchiectomy by open technique to prevent herniation. the reproductive tract of the doe is characterized by two uterine horns that are connected to the vagina by separate cervices (bicornuate uterus). a common tube, the urogenital sinus or vestibulum, is present where the urethra enters the vagina. the placenta is hemochorial, and maternal blood flows into sinus-like spaces where the transfer of nutrients and other substances to the fetal circulation occurs (jones and hunt, ) . inguinal pouches are located lateral to the genitalia in both sexes. the pouches are blind and contain scent glands that produce white to brown secretions that may accumulate in the pouch. the metabolic rate of endotherms is generally related to the body surface area. including the ears, the rabbit has a relatively low metabolic rate (mr); however, if the surface area of the ears is discounted, the mr of the rabbit is similar to that of other endotherms. neonatal rabbits have an amount of body fat comparable to that of the human infant ( % of body weight) (cornblath and schwartz, ) . the neonatal rabbit is essentially an ectotherm until about day (gelineo, ) . the glucose reserves of the neonatal rabbit are quickly depleted, usually within about h after birth (shelley, ) . the fasting neonatal rabbit quickly becomes hypoglycemic and ketotic (callikan and girard, ) . the normal rectal temperature of the adult new zealand white rabbit at rest is approximately . - . °c (ruckebusch et al., ) . the ears serve an important thermoregulatory function. because they have a large surface area and are highly vascular with an extensive arteriovenous anastomotic system, the ears help the rabbit sense laboratory animal medicine and respond to cold versus warm temperatures (kluger et al., ) . in addition, the ears serve as a countercurrent heat-exchange system to help adjust body temperature. early studies found that the body of the adult rabbit ( kg body weight) consists of greater than % water ( %), with a half-time turnover of about . days and a loss of about ml daily (richmond et al., ) . the amount of water ingested varies with the amount and type of feed consumed and the environmental temperature. in general, rabbits will drink more water when consuming dry, pelleted feed than when consuming foodstuffs high in moisture, such as fresh greens. conversely, rabbits deprived of water will decrease food consumption. after days of complete water deprivation, food intake falls to less than % of normal (cizek, ) . normal values for various systems and parameters are provided as a general indication for these values in the rabbit. it is important to recognize, however, that most of these values have been obtained through the study of adult new zealand white rabbits. as with any experiment, values can vary significantly between breeds, laboratories, methods of sampling and measurement, and individual rabbits due to age, sex, breed, health, handling, and husbandry (hewitt et al., ; lidena and trautschold, ; mitruka and rawnsley, ; wolford et al., ; yu et al., ) . for this reason, individual laboratories should strive to establish their own normal values, whenever possible. values for hematologic parameters are shown in table . . these values represent those typical of adult new zealand white rabbits. in general, males have slightly greater hematocrit and hemoglobin values than females (mitruka and rawnsley, ) . anisocytosis is normal and accounts for variation in reported values for red blood cell diameter (sanderson and phillips, ) . reticulocyte values are usually between % and % in healthy rabbits (corash et al., ) . the neutrophil of the rabbit is sometimes referred to as a 'pseudoeosinophil' or 'heterophil,' due to the presence of red-staining granules in the cytoplasm. the heterophil ( - mm in diameter) is, however, smaller than the eosinophil ( - mm in diameter) (sanderson and phillips, ) . in addition, the red granules of the heterophil are smaller than the red granules of the eosinophil. the nucleus of the eosinophil may be either bilobed or horseshoe-shaped. as mentioned earlier, chemistry values can vary because of a number of factors. for this reason, each laboratory should establish its own normal values. aspartate aminotransferase (ast) is present in the liver, heart, skeletal muscle, kidney, and pancreas. collection of blood samples in rabbits by decapitation, cardiac puncture, or aortic incision, or the use of restraint that causes exertion will elevate ast levels due to muscle damage (lidena and trautschold, ) . similarly, levels of creatinine kinase are sensitive to muscle damage since that enzyme is present in the skeletal muscle, brain, and heart (lidena and trautschold, ; mitruka and rawnsley, ) . although most mammals have two isoenzymes (intestinal and a liver/kidney/bone form) of alkaline phosphatase (ap), rabbits are unique in having three forms of ap, including an intestinal form and two forms that are both present in the liver and the kidney (noguchi and yamashita, ) . values for blood and serum chemistry are shown in table . . cardiovascular and respiratory functions are often altered with experimental manipulation, anesthesia, or disease. normal values for these parameters and other miscellaneous biologic characteristics of the rabbit are listed in table . . values obtained from the following sources: burns and delannoy ( ) , gillett ( ) , kabata et al. ( ) , mitruka and rawnsley ( ), and woolford et al. ( ) . laboratory animal medicine rabbits are strictly herbivorous with a preferred diet of herbage that is low in fiber and high in protein and soluble carbohydrate (cheeke, ; cheeke, ) . rabbits will generally accept a pelleted feed more readily than one in meal form. when a meal diet is needed, a period of adjustment should be allowed for the rabbits to accommodate to the new diet. examples of adequate diets are shown in table . . the requirement for fiber in the diet of rabbits has been reviewed (gidenne, ) . fiber is especially important in the early postweaning period when low fiber intake is associated with an increase in digestive disorders (gidenne, ) . the exact nutrient requirements for individual rabbits vary with age, reproductive status, and health of the animal. on occasion, the need arises for use of highly purified diets. a suggested purified diet has been described elsewhere (subcommittee on rabbit nutrition, ) . it should be noted that overfeeding of values obtained from the following sources: burns and delannoy ( ) , fox ( ) , gillett ( ) , kraus et al. ( ), and loeb and quimby ( ) . values obtained from the following sources: barzago et al. ( ) , curiel et al. ( ) , gillett ( ) , kozma et al. ( ) , sanford and colby ( ) , suckow and douglas ( ), and zurovsky et al. ( ) . laboratory animal medicine laboratory rabbits resulting in obesity is common, but can be prevented by either reducing the amount of feed or by providing a low-energy, high-fiber maintenance diet (donnelly, ) . as mentioned earlier, rabbits engage in cecotrophy, and by doing so supplement their supply of protein and Β vitamins (carabaño et al., ; gidenne et al., ) . rabbits fed a diet high in fiber ingest a greater quantity of cecotropes than those on a lower fiber diet (fekete and bokori, ) . unlike most other species, both calcium absorption in the small intestine and serum calcium levels increase in proportion to the amount of calcium in the diet (cheeke, ) . prolonged feeding of diets high in calcium, such as those with a high level of alfalfa meal, can result in renal disease. consumption of diets containing excessive vitamin d can result in calcification of soft tissues, including the liver, kidney, vasculature, and muscles (besch-williford et al., ; lebas, ) . diets that are either too high or too low in vitamin a can result in reproductive dysfunction and congenital hydrocephalus (cheeke, ; digiacomo et al., ) . the exact requirement for vitamin a in the rabbit has not been determined; however, a level of - , iu/kg of diet is generally adequate (lebas, ) . vitamin e deficiency has been associated with infertility, muscular dystrophy, fetal death, neonatal death, and colobomatous microphthalmos in rabbits (lebas, ; nielsen and carlton, ; ringler and abrams, ; ringler and abrams, ) . mcdowell ( ) suggested that serum vitamin e levels of less than . μg/ml are indicative of hypovitaminosis e. relative to other species, rabbits have a high water intake. in general, daily water intake is approximately ml/kg of body weight. consumption of water is influenced by environmental temperature, disease states, and feed composition and intake (cizek, ; tschudin et al., ) . consumption of diets high in dry matter results in increased water intake (tschudin et al., ) . water consumption also increases with food deprivation. rabbits are social animals and attempts at group housing often meet with success, although mature males will fight and can inflict serious injury on one another (love, ; podberscek et al., ; whary et al., ) . group-penned female rabbits allowed to choose between single or paired housing prefer being in the same cage with other rabbits (huls et al., ) . in general, rabbits are timid and nonaggressive. some animals will display defensive behavior, typically characterized by thumping the cage floor with the rear feet, biting, and charging toward the front of the cage when opened. laboratory-housed rabbits demonstrate diurnal behavior, in contrast to the nocturnal pattern exhibited by wild rabbits (jilge, ) . the ethogram of the laboratory rabbit has been described (chu et al., ; gunn and morton, ) . the most common behaviors of individually housed rabbits included lie alert, doze, groom, sleep, and eat. individually housed rabbits were inactive the majority of the time (gunn and morton, ) . individually housed female rabbits showed an increase in abnormal behaviors compared to pair-housed rabbits (chu et al., ) . rabbits housed in pairs in double-wide cages locomoted more than individually housed rabbits (chu et al., ). the age of puberty varies with the breed of rabbit. puberty generally occurs at - months of age in small breeds, - months in medium breeds, and - months in large breeds (donnelly, ) . female new zealand white rabbits reach maturity at months of age and males at - months. the breeding life of a doe typically lasts approximately - years, although some remain productive for up to or years. in later years, litter sizes usually diminish. in comparison, most bucks will remain reproductively useful for an average of - years. because does often will engage in reproductive behavior before being able to ovulate, it is advisable not to breed does until they are fully grown. does do not have a distinct estrous cycle, but rather demonstrate a rhythm with respect to receptivity to the buck. receptivity is punctuated by periods ( - days every - days) of anestrus and seasonal variations in reproductive performance (hafez, ) . during periods of receptivity, the vulva of the doe usually becomes swollen, moist, and dark pink or red. receptivity of the doe is usually signaled by lordosis in response to the buck's attempt to mount, vulvar changes as described above, restlessness, and rubbing of the chin on the hutch or cage (donnelly, ) . vaginal cytology is generally not useful for determination of estrus or receptivity in the rabbit. typically, the doe is brought to the buck's cage for breeding, since the doe can be very territorial and may attack the male in her own quarters. a period of - min is usually sufficient to determine compatibility of the doe and buck. if receptive, the doe will lie in the mating position and raise her hindquarters to allow copulation. if fighting or lack of breeding is observed, the doe may be tried with another buck. a single buck is usually sufficient to service - does. ovulation is induced and occurs approximately - h after copulation (donnelly, ) . up to % of does fail to ovulate following copulation. ovulation can also be induced by administration of luteinizing hormone (kennelly and foote, ) , human chorionic gonadotropin (williams et al., ) , or gonadotropic releasing hormone (foote and simkin, ) . does may be bred immediately after kindling; however, most breeders delay until after the kits have been weaned. success at postpartum breeding varies, but one can produce a large number of kits in a relatively short time period by foster nursing the young and rebreeding the doe immediately. while conventional breeding, nursing, and weaning schedules allow for only litters per year, early postpartum breeding allows for up to litters per year. pregnancy can often be confirmed as early as day of gestation by palpation of the fetuses within the uterus. radiographic procedures permit pregnancy determination as early as day . conception rates have been observed to have an inverse relationship with ambient temperature but not light cycle. gestation in rabbits usually lasts for - days (donnelly, ) . does beyond - weeks of gestation will usually refuse a buck. does begin hair pulling and nest building during the last - days of gestation (donnelly, ) . a nesting box with shredded paper or other soft material such as straw should be provided to the doe several days prior to the expected kindling (parturition) date. the doe will usually line the box with her own hair. the nesting box should not be placed in the corner of the cage where the individual doe has been observed to urinate. pseudopregnancy is common in rabbits and can follow a variety of stimuli, including mounting by other does, sterile matings by bucks, administration of luteinizing hormone, or the presence of bucks nearby. in such circumstances, ovulation is followed by a persistent corpus luteum that lasts - days. the corpus luteum or corpora lutea secretes progesterone during this time, causing the uterus and mammae to enlarge. the doe may have the appearance of a normally pregnant rabbit. toward the end of pseudopregnancy, many does will begin to pull hair as part of ritual nest-building behavior. the process of parturition is referred to as 'kindling' when it relates to rabbits. kindling normally occurs during the early morning hours and takes approximately - min. impending kindling is often signaled by nest building and decreased food consumption during the preceding - days. both anterior and breech presentations are normal in the rabbit. fetuses retained beyond days generally die and may harm future reproductive ability of the doe if not expelled. the average number of kits born is seven to nine per litter, although smaller litters and litters of up to kits are not uncommon. breed, parity, nutritional status, and environmental factors influence litter size. polish rabbits usually have fewer than four kits per litter; dutch or flemish giant, four to five; and new zealand white, eight to ten. after the young have been cleaned following parturition, the doe typically consumes the placenta. cannibalism of the young by the doe sometimes occurs and may be related to environmental or hereditary factors or due to environmental stressors. does usually have either four or five pairs of nipples, whereas bucks have none. during the last week of pregnancy, marked development of the mammary gland occurs. the doe normally nurses the kits once daily for several minutes, usually in the early morning or in the evening, regardless of how many kits are present or laboratory animal medicine how many times they attempt to suckle. milk yield is normally between and g/day. during the first week of life, kits consume - g of milk per day. milk intake increases gradually to a maximum of g/day between and days of age (gidenne et al., ) . maximum output occurs at weeks following kindling and then declines during the fourth week. rabbit milk contains approximately . % protein, % fat, % lactose, and . % minerals. nursing may last - weeks. kits may begin consuming solid food by weeks of age, with weaning generally occurring by - weeks of age. the facilities present in most modern research animal facilities would be suitable for housing rabbits. general construction should include adequate heating, ventilation, and air conditioning to house rabbits at appropriate temperature and humidity. in addition, lighting should be adequate to allow easy visualization of the rabbits. surfaces, such as the floors, walls, and ceilings, should be easily sanitizable (national research council, ) . rabbit cages should provide a safe environment with easy access to food and water. adults can be caged individually or in compatible groups and should have sufficient floor space to lie down and stretch out. in the united states, minimum cage sizes are determined by the animal welfare act (awa) and the guide for the care and use of laboratory animals (guide). in both cases, sizes vary with the weight of the animal. currently, the awa regulations and the guide require . ft of floor space and in of cage height for rabbits weighing - kg (national research council, ) . cages should be constructed of durable materials that will resist corrosion and harsh detergents and disinfectants used in cleaning. consequently, in the research environment, rabbit cages are most often constructed of stainless steel or plastics. rabbits are usually housed in cages with mesh or slatted floors to permit urine and feces to drop through into a catch pan. mesh floors with catch pans do not prevent rabbits from engaging in the normal practice of coprophagy. information on environmental enrichment of laboratory rabbits has been published (baumans, ) . the behavior of rabbits in conventional cages was compared to that of rabbits provided with enriched cages that contained shelter, a shelf, and increased vertical space. rabbits in conventional cages were more restless, groomed excessively, exhibited more bar-gnawing, and were more timid than those housed in enriched cages (hansen and berthelsen, ) . indeed, fecal glucocorticoid levels in rabbits declined when they were provided with a wooden structure for resting and gnawing (buijs et al., ) . rabbits will play with objects placed in their cages. huls et al. ( ) noted that rabbits would use wooden sticks, wooden rings, and brass wire balls as toys. rabbits provided with objects (toys) spent significantly more time chewing than rabbits without toys (poggiagliolmi et al., ) . female rabbits can also be housed in compatible pairs or groups. singly housed female rabbits exhibited more abnormal behaviors compared to pair housed rabbits (chu et al., ) . group housing of unfamiliar males is not recommended because of the likelihood of fighting and injury. rabbits are optimally housed in cooler room temperatures than most other common species of laboratory animals. the guide recommends that temperatures in rabbit rooms be maintained between and °f. no specific illumination requirements for rabbits have been described. it is common practice to provide rabbits with - h of light in the light-dark cycle. in breeding colonies, females should be provided with - h of light. rabbits are easily startled by sudden, loud noises. for this reason, they should not be housed near noisy species such as dogs or monkeys, nor should they be housed near noise-generating operations such as the cage-wash area. catch pans should be cleaned as often as necessary to prevent the formation of ammonia. cages are generally sanitized on at least a weekly basis. rabbit urine contains large amounts of protein and minerals, and often forms deposits on cages and catch pans. it is common practice to soak equipment having urine deposits in acid washes to remove the scale before washing. ammonia production in rabbit rooms can be a significant problem; therefore, rabbit rooms should be ventilated at - air changes per hour (national research council, ) . it is also important to change excreta pans often to prevent the buildup of ammonia. etiology pasteurella multocida is a gram-negative nonmotile coccobacillus that causes pasteurellosis, also known as 'snuffles', the primary respiratory disease affecting domestic rabbits (deeb and digiacomo, ; guo et al., ) . historically, serogroup a isolates have laboratory animal medicine been associated with pneumonic and septicemic pasteurellosis in laboratory rabbits; however, capsular type a is also isolated from rabbits that appear clinically healthy (confer et al., ; el tayeb et al., ) . clinical signs pasteurella multocida infection is often subclinical, but pasteurellosis may cause fever, coughing, dyspnea, rhinitis (nasal discharge (serous to mucopurulent), sneezing, and upper airway stentor), pneumonia, otitis, septicemia, meningitis, abscesses (of viscera and subcutaneous sites), and death (al-lebban et al., ; confer et al., ; franco and cronin, ; guo et al., ; suckow et al., ; wilkie et al., ) . pasteurellosis may also be associated with pericarditis, pleuritis, sinusitis, dacryocystitis, conjunctivitis, iritis/ uveitis, phlegmon, mastitis, endometritis, pyometra, salpingitis, and orchitis (deeb and digiacomo, ; ferreira et al., ; stahel et al., ; williams, ) . epizootiology p. multocida can be endemic in rabbitries and is carried in the rabbit's nasal cavity (confer et al., ; deeb et al., ; digiacomo et al., ; suckow et al., ) . transmission is by direct contact between rabbits (wilkie et al., ) . coinfection with bordetella bronchiseptica may be observed in clinically affected rabbits (deeb et al., ) . stress-related factors associated with pasteurellosis include crowded or unsanitary conditions, transportation, and high ammonia concentrations in the air (confer et al., ) . previous studies reported a high prevalence of p. multocida infection (jaslow et al., ) . colonization in immature rabbits occurs more commonly in the sinuses followed by the trachea, middle ears, and lungs (glass and beasley, ) . similar to cats and dogs, rabbits may transmit p. multocida infection to humans (per et al., ; silberfein et al., ) . a study utilizing repetitive extragenic palindromic pcr (rep-pcr) and sequencing determined that % of the isolates were characterized as p. multocida subsp. multocida, % as p. multocida subsp. septica, % as atypical subspecies of p. multocida, % as p. canis, and % as an unknown species of the family pasteurellaceae (stahel et al., ) . the pathogenesis of p. multocida has been reviewed (wilkie et al., ) . the ptfa gene, encoding a type fimbrial subunit and involved in bacterial fixation on the surface of epithelial cells, may be highly prevalent in p. multocida isolates from rabbits (ferreira et al., ) . the p. multocida toxin is a major virulence factor in atrophic rhinitis of rabbits and acts by causing constitutive activation of g proteins frymus et al., ; orth et al., ; suckow et al., ) . pathology the specific pathologic findings will vary with the site of infection, but the underlying host response is characterized by acute or chronic suppurative inflammation with the infiltration of large numbers of neutrophils. rhinitis and sinusitis are accompanied by a mucopurulent nasal exudate. neutrophil infiltration of the tissues is extensive. the nasal passages are edematous, inflamed, and congested, and there may be mucosal ulcerations. the turbinate bones may atrophy digiacomo et al., ) . purulent conjunctivitis may be present. pneumonia is primarily cranioventral in distribution. the lungs can exhibit consolidation, atelectasis, and abscess formation. a purulent to fibrinopurulent exudate is evident, and there may be areas of hemorrhage and necrosis. in some rabbits, fibrinopurulent pleuritis and pericarditis are prominent features (glavits and magyar, ) . this is probably due to elaboration of a heat-labile toxin in some strains of the bacteria . acute hepatic necrosis and splenic lymphoid atrophy are also seen in association with the pleuritis and pneumonia induced by toxigenic strains. otitis media is characterized by a suppurative exudate with goblet cell proliferation and lymphocytic and plasma cell infiltration. in female rabbits with genital tract infections, the uterus may be enlarged and dilated. in the early stages of infection, the exudate is watery; later it thickens and is cream-colored. the exudate contains numerous neutrophils. focal endometrial ulceration can be found (johnson and wolf, ) . in the male, the testes are enlarged and may contain abscesses. systemic and visceral abscesses are characterized by a necrotic center, an infiltrate made up of polymorphonuclear neutrophils, and a fibrous capsule. septicemia may only present as congestion and petechial hemorrhages in many organs. severe pleuritis with accumulation of fibrinopurulent exudate in the thoracic cavity, serous rhinitis and tracheitis, acute hepatitis with necrotic foci in the parenchyma, and atrophy of lymphoid organs and tissues have been observed after experimental p. multocida infection in rabbits (glavits and magyar, ) . diagnosis sterile swabs can be used to collect samples from the nares or nasal cavity of rabbits for culture (ferreira et al., ; jaslow et al., ) . nasal lavage can also be used as a culture sample to isolate pasteurella (suckow et al., ) . p. multocida isolates can be classified into five serogroups based on capsular antigens (a, b, d, e, and f) and into serotypes based on somatic lps antigens (adler et al., ; liu et al., b; manning, ) . biochemical characterization of isolates may show high heterogeneity; however, rep-pcr and phylogenetic analysis using s ribosomal rna and rpob genes can be used for precise characterization of rabbit isolates (stahel et al., ) . classification of p. multocida into subspecies and/or by virulence profiles is useful for epidemiological investigations (ferreira et al., ; stahel et al., ) . random amplified polymorphic laboratory animal medicine dna pcr (rapd-pcr) has also been used to subtype rabbit p. multocida isolates (al-haddawi et al., ; dabo et al., ; williams et al., ) . pcr can detect capsule biosynthesis genes cap a, b, d, e, and f as well as virulence-related genes (ferreira et al., ) . serological tests can be used to detect antibodies against p. multocida (deeb et al., ; delong et al., ; digiacomo et al., ; glass and beasley, ; lukas et al., ) . differential diagnoses if radiographs reveal an internal mass associated with p. multocida infection, the differential diagnoses should include abscess, granuloma, neoplasia, and parasitic cyst (franco and cronin, ) . treatment, prevention, and control previous studies have investigated the use of vaccines to protect rabbits against p. multocida infection (confer et al., ) . immunization of rabbits with inactivated heat-labile p. multocida toxin or a commercial swine p. multocida bacterin-toxoid conferred protective immunity against challenge with the p. multocida heat-labile toxin (suckow, ; suckow et al., ) . a vaccine administered intranasally stimulated immunity against experimental pneumonic pasteurellosis and significantly reduced nasal bacterial counts (confer et al., ) . oral immunization of rabbits with a p. multocida thiocyanate extract (pte) in microparticles was immunogenic and significantly reduced the colony-forming units of homologous p. multocida recovered from the lungs and nasopharynx (suckow et al., ) . protective immunity to a heterologous strain of p. multocida can be achieved by vaccinating rabbits with pte via the subcutaneous route (suckow et al., ) . a p. multocida bacterin known as bunnyvac is currently licensed by the usda and is intended to be effective in preventing death and limiting disease due to pasteurella in rabbits. bunnyvac is manufactured by colorado serum company and distributed by pan american veterinary laboratories (http://pavlab. com/). control of pasteurellosis in rabbitries entails testing and culling animals that are positive for pasteurella spp. (ferreira et al., ) . furthermore, rabbits free of pasteurella and other infectious agents can be obtained by enrofloxacin treatment and through cesarean section or hysterectomy rederivation (pleasants, ; suckow et al., ; syukuda, ) . commercial suppliers of laboratory rabbits tend to exclude pasteurella from their colonies. treatment with antibiotics should be based on culture and sensitivity. antibiotic treatment of affected rabbits can alleviate clinical signs or delay disease progression but may not eradicate the disease (el tayeb et al., ; ferreira et al., ) . antibiotic treatment may suppress virulence gene expression without complete elimination of p. multocida . internal abscesses may not be treatable using antibiotics (franco and cronin, ) . penicillin therapy does not seem to be effective against pasteurella infection and may also lead to diarrhea and clostridium difficile colitis in rabbits (jaslow et al., ; rehg and lu, ) . one study from brazil determined that all tested strains were sensitive to ceftiofur, florfenicol, norfloxacin, enrofloxacin, ciprofloxacin, tetracycline, and doxycycline (ferreira et al., ) . other studies also indicate that fluoroquinolones are useful for the treatment of p. multocida infection in rabbits (abo-el-sooud and goudah, ; broome and brooks, ; franco and cronin, ; hanan et al., ; okewole and olubunmi, ) . oral ciprofloxacin ( mg/kg per day for days) has been used in rabbits (hanan et al., ) . research complications pasteurellosis can cause considerable economic losses (el tayeb et al., ; ferreira et al., ; stahel et al., ) and has the potential to affect different types of research studies using rabbits due to the multisystemic nature of the disease, and the possibility of high morbidity and mortality. therefore, pasteurella should be excluded from laboratory rabbit colonies. the class clostridia belongs to the phylum firmicutes (yutin and galperin, ) . recent genomic analyses suggest assigning some clostridium species that fall outside the family clostridiaceae into new genera. the genera tyzzerella, erysipelatoclostridium, and peptoclostridium have been proposed for c. piliforme, c. spiroforme, and c. difficile, respectively (yutin and galperin, ) . etiology c. piliforme is a pleomorphic, gramnegative, spore-forming, motile, obligate intracellular rod-shaped bacterium that causes tyzzer's disease and infects various animals including mice, nonhuman primates, gerbils, rats, rabbits, and others (allen et al., ; ganaway et al., ; pritt et al., ) . infection has also been reported in a human patient with human immunodeficiency virus- (smith et al., ) . phylogenetic analyses determined that microorganisms identified as c. piliforme form three clusters within a single clade and that the nearest related distinguishable species is c. colinum (feldman et al., ) . clinical signs the first reported outbreaks in laboratory rabbits described profuse watery to mucoid diarrhea usually followed by death in - h in -to -week old rabbits (allen et al., ) . rabbits in affected litters usually died within a week after the first fatality (allen et al., ) . the dams of affected litters occasionally died after a diarrheal disease that was more protracted than that of the offspring (allen et al., ) . these outbreaks lasted for - months and affected multiple rabbit rooms. c. piliforme infection may also be subclinical and transient as immunocompetent hosts may clear the infection (ganaway et al., ; pritt et al., ) . weanling rabbits with the acute form of tyzzer's disease exhibit diarrhea, listlessness, anorexia, and dehydration usually followed by death within h (cutlip et al., ) . the mortality rate in clinically affected rabbits was estimated to be - % (cutlip et al., ) . anorexia and stunting were observed in chronic cases associated with intestinal stenosis (cutlip et al., ) . acute and chronic tyzzer's disease types have been described in rabbits; however, large numbers of 'attaching' escherichia coli were recovered from the cecum of most rabbits (prescott, ) . epizootiology the vegetative cell is the active stage responsible for the disease and depends on the intracellular environment (ganaway, ) . therefore, the spore, a resistant stage, appears to be the essential element in the transmission of tyzzer's disease (ganaway, ; ganaway et al., ) . contact with soiled bedding or diseased rabbits have been used experimentally to transmit the disease to other rabbits (allen et al., ) . it is possible that subclinically infected rabbits (carriers) may introduce the organism into a colony (allen et al., ; pritt et al., ) . in mice, increased susceptibility to infection has been associated with stress (allen et al., ) . furthermore, treatment with cyclophosphamide, cortisone, and prednisolone has been used experimentally to reproduce the disease in animals, suggesting that immunosuppression plays a role in pathogenesis (allen et al., ; cutlip et al., ; pritt et al., ) . animals stressed by poor environmental conditions including overcrowding and extreme temperatures can develop the disease (cutlip et al., ; wobeser et al., ). significant modifications of the intestinal flora and an impaired immune system may play a role in pathogenesis (licois, ) . c. piliforme may be transported from the intestine to the liver through the portal circulation and to the heart through the lymphatics (allen et al., ) . some c. piliforme isolates can induce cytopathic effects on cell cultures, and in vivo, concomitant infection with other enteric pathogens such as e. coli may contribute to the severity of the disease (prescott, ; riley et al., ) . pathology lesions can be found in the distal ileum, cecum, proximal colon, liver, and heart (allen et al., ) . intestinal lesions are common, and histologically are characterized by necrosis of the mucosa and edema of the submucosa and serosa (allen et al., ) . bacilli appear as bundles of parallel rods or as criss crossed sticks in the cytoplasm of epithelial cells distributed from the surface of the mucosa to the base of the glands (allen et al., ) . the lesions in the liver are punctate areas of parenchymal necrosis that appear grossly as white spots, usually ≤ mm in diameter. large numbers of bacilli are found in the cytoplasm of cells in the zone of transition between the necrotic lesion and the healthy parenchyma (allen et al., ) . myocardial lesions appear as white streaks . - mm wide and - mm long extending from the region of the left interventricular groove laterally across the left ventricle (allen et al., ) . in the myocardium, bacilli may be noted in partially degenerated and normal looking cells at the sharply delineated borders of the lesions (allen et al., ) . diagnosis c. piliforme cannot be cultured in artificial (cell-free) media making its diagnosis difficult (allen et al., ; cutlip et al., ; ganaway et al., ; niepceron and licois, ) . other bacteria, including e. coli, have been isolated from the liver of diseased rabbits and are considered secondary invaders (allen et al., ; cutlip et al., ) . the isolation of the tyzzer's agent using liver extract agar has been described (kanazawa and imai, ) . c. piliforme can be grown in primary monolayer cultures of adult mouse hepatocytes, in mouse fibroblasts, in rat hepatocytes, and in embryonated eggs (craigie, ; duncan et al., ; ganaway et al., ; kawamura et al., ; pritt et al., ; riley et al., ) . serology for c. piliforme is commonly used for surveillance of laboratory animals because it is rapid and inexpensive (pritt et al., ) . immunofluorescence assay (ifa) and multiplexed fluorometric immunoassay (mfia) have been utilized (pritt et al., ) . in addition, c. piliforme pcr assays have been developed (feldman et al., ; gao et al., ; niepceron and licois, ; pritt et al., ) . clostridium piliforme seropositive rabbits may be negative for the organism by pcr and histopathological evaluation (pritt et al., ) . therefore, positive serological findings are not sufficient for a definitive diagnosis of c. piliforme infection and pcr testing and/or histopathology should be used for confirmation (pritt et al., ) . definitive diagnosis is based on identification of typical gross lesions and histological demonstration of intracellular c. piliforme at the periphery of the necrotic foci (niepceron and licois, ; pritt et al., ) . giemsa solution (ph ), warthin-starry silver method, levaditi silver method, and the periodic acid-schiff (pas) reaction have been used to demonstrate c. piliforme (allen et al., ; cutlip et al., ) . different morphologic forms of c. piliforme can be observed microscopically (allen et al., ; ganaway et al., ) . differential diagnoses clinically, other diarrheal diseases of rabbits can be included in the differential diagnoses. grossly, the multifocal white areas on the liver could be from eimeria stiedae infection (hepatic coccidiosis). treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown c. piliforme status into a colony. minimize stress-related factors especially in young animals. good husbandry practices including regular bedding changes and disinfection laboratory animal medicine should decrease the likelihood of spreading c. piliforme in a colony. in one report, a tyzzer's disease outbreak was observed - days after rabbits were weaned and transferred to a facility in which the temperature fluctuated from to °c. the outbreak was controlled by transferring weanling rabbits to a building maintained at the same temperature as the breeder house ( - °c) (cutlip et al., ) . spores treated with heat ( or °c) or with either peracetic acid ( %) in a wetting agent (sodium alkylarylsulfonate) or sodium hypochlorite solution ( . %) for min lose infectivity (ganaway, ) . however, spores do not lose infectivity when treated with a phenolic germicidal agent, ethanol, or quaternary ammonium compounds (containing % or % benzalkonium chloride) (ganaway, ) . sodium hypochlorite solution ( . %) has been recommended as a surface disinfectant in animal facilities for prevention and control of tyzzer's disease (ganaway, ) . the sensitivity of c. piliforme to antibiotics has been investigated (kanazawa and imai, ) . in one study, none of the antibacterials tested were completely inhibitory (ganaway et al., ) . group treatment of rabbits with tetracyclines in the drinking water and food was effective in lowering the incidence of diarrhea and death (prescott, ) . research complications the high morbidity and mortality associated with tyzzer's disease can affect the overall population of rabbits in a colony thereby decreasing the number of rabbits suitable or available for experimentation. in addition, research studies involving experimental infection with enteric pathogens in rabbits may be confounded by c. piliforme-associated intestinal pathology. enterotoxemia refers to conditions of the bowel caused by toxigenic clostridia (carman and evans, ) . diagnosis of enterotoxemia should be based on culture of a toxigenic clostridium and demonstration of the toxin from the intestinal contents of the diseased animal (carman and evans, ; songer, ) . i. clostridium spiroforme etiology c. spiroforme is a gram-positive, sporebearing, helically coiled, semicircular, anaerobic bacterium that can produce iota toxin (borriello and carmen, ; carman and borriello, ; peeters et al., ) . the disease caused by c. spiroforme is known as 'iota enterotoxemia' (keel and songer, ; peeters et al., ) . clinical signs diarrhea, fecal soiling of the perineum, and cyanosis may be observed (carman and borriello, ) . diarrhea may be peracute and may lead to 'spontaneous' death or a moribund state (carman and borriello, ) . epizootiology c. spiroforme is thought to be acquired from the environment (carman and evans, ; songer, ) . weaning is associated with spontaneous disease and administration of antibiotics can also induce the disease (borriello and carmen, ; carman and borriello, ) . a study determined that disease results from exposure of weanling rabbits to c. spiroforme and also from exposure of adult rabbits with a disrupted gut flora (due to clindamycin treatment) to c. spiroforme suggesting that this bacterium is not a normal component of the rabbit gut flora (carman and borriello, ) . c. spiroforme-mediated diarrhea may be favored by maldigestion initiated by infectious agents and/or nutritional factors (peeters et al., ) . other clostridia, e. coli (epec), viruses, and parasites, may coinfect rabbits (peeters et al., ) . the iota toxin of c. spiroforme binds the same host cell receptor as the iota toxin of c. perfringens and the binary toxin of c. difficile (papatheodorou et al., ) . poor hygiene, stress, and diet can influence pathogenesis of the disease (bain et al., ; songer, ) . pathology grossly, ceca may be enlarged with gas, may exhibit serosal hemorrhages, and have liquid contents (carman and borriello, ) . cecal contents may be stained with blood (peeters et al., ) . c. spiroforme is mainly associated with lesions in the cecum, but may also involve lesions in the proximal colon and distal ileum (keel and songer, ) . mucosal necrosis can be observed microscopically (keel and songer, ) . the mucosa of the ileum, cecum, and colon may be denuded. cellular debris, red blood cells, and fibrin may be found in the intestinal lumen. polymorphonuclear cell infiltration and edema can be found in the lamina propria and submucosa. epithelial degeneration and dilation were found in the renal tubules of some rabbits (peeters et al., ) . diagnosis gram staining of smears prepared from intestinal contents can be used to detect c. spiroforme (bain et al., ) . clostridial culture and toxin detection assays have been described (agnoletti et al., ; bain et al., ; borriello and carmen, ; peeters et al., ) . c. spiroforme can be isolated from the intestinal contents of rabbits by high-speed centrifugation (holmes et al., ) . pcr assays for c. spiroforme and the iota toxin (binary toxin) have been developed (drigo et al., ) . differential diagnoses the differential diagnoses should include other clostridia, e. coli, viruses, and parasites (peeters et al., ) . treatment, prevention, and control the iota toxin from c. spiroforme is neutralized by serum prepared against the iota toxin of c. perfringens type e (borriello and carmen, ; carman and borriello, ; songer, ) . prevention, via reduction of risk factors and prudent use of antibiotics, is probably more important laboratory animal medicine than treatment (agnoletti et al., ) . cholestyramine has been used to prevent experimental enterotoxemia induced by clindamycin in rabbits (lipman et al., ) . fecal flora transplants using nonpathogenic c. spiroforme or c. difficile have been suggested for competitive inhibition of toxigenic strains (carman and evans, ) . no commercial vaccines are available for rabbits; however, vaccination of weanling rabbits with a toxoid imparted protection to intraperitoneal challenge with iota toxin (songer, ) . administration of antibiotics and change in diet are usually the treatment for c. spiroforme infections (songer, ) . the antibiotic susceptibility of c. spiroforme has been investigated (agnoletti et al., ; carman and wilkins, ) . c. spiroforme can have a wide range of resistance to antimicrobial classes used in rabbit therapy (agnoletti et al., ) . feeding fresh meadow hay has been suggested (bain et al., ) . research complications the mortality due to enterotoxemia caused by c. spiroforme would be disruptive to ongoing studies. no other complications have been reported. ii. clostridium difficile etiology c. difficile is a gram-positive, sporeforming, anaerobic bacillus commonly associated with diarrhea and colitis in humans and animals (keel and songer, ) . clinical signs c. difficile infection may be associated with anorexia, depression, diarrhea, fecal-staining of the perineum, decreased fecal output, abdominal distention, and death (perkins et al., ; rehg and lu, ) . peracute death, without clinical signs, is also a common presentation in rabbits (keel and songer, ; perkins et al., ) . epizootiology the spread of c. difficile involves carrier animals that do not show clinical signs of disease (keel and songer, ) . the carrier state may depend on the age of the individual (keel and songer, ) . c. difficile is thought to be acquired from the environment due to persistent contamination with spores (keel and songer, ) . disease is associated with antibiotic treatment but can also develop spontaneously (without antibiotic treatment) (perkins et al., ; rehg and lu, ) . the disease may also occur after stressful events such as weaning, sudden dietary changes, lactation, kindling, and illness (perkins et al., ) . rabbits that have been recently weaned are the most susceptible (perkins et al., ) . newborn rabbits are resistant to c. difficile disease possibly due to the lack of receptors for the toxins (keel and songer, ) . similar to c. spiroforme, the pathogenesis is associated with disruption of the gut flora and with colonization and proliferation of toxigenic clostridium. pathology grossly, a fluid-filled cecum and colon may be found on necropsy (rehg and lu, ) . spontaneous disease in rabbits is associated with lesions in the small intestine, most commonly in the ileum (keel and songer, ) . in one study, the small intestine was distended with fluid and the cecum was distended with chyme (perkins et al., ) . c. difficile is also associated with hemorrhagic typhlitis in hares (dabard et al., ) . c. difficile causes severe jejunal lesions in rabbits, but cecal lesions may occur (keel and songer, ; perkins et al., ) . mural hemorrhages, mucosal necrosis, and submucosal edema have been observed (perkins et al., ) . toxins a (enterotoxin) and b (cytotoxin) act synergistically and are essential virulence factors of c. difficile that enter the cells through receptor-mediated endocytosis (keel and songer, ) . toxins a and b disrupt the actin cytoskeleton by disrupting rho-subtype intracellular signaling molecules that affect cellular function (keel and songer, ) . inflammation and neurogenic stimuli also are involved in the pathogenesis of c. difficile disease (keel and songer, ) . in addition to toxins a and b, some c. difficile strains produce an actinspecific adp-ribosyltransferase or binary toxin (stubbs et al., ) . diagnosis c. difficile isolation and toxin assays have been described (keel and songer, ; perkins et al., ; rehg and lu, ) . c. difficile selective agar is commercially available. the tissue culture cytotoxin assay for c. difficile toxin b is considered the 'gold standard' (belanger et al., ) . c. difficile toxin b can be neutralized with c. sordelli antiserum, but not with c. spiroforme antiserum (perkins et al., ) . commercially available enzyme immunoassays to detect c. difficile toxin(s) have been used to diagnose rabbit cases (garcia et al., ; perkins et al., ) . pcr assays have been developed (belanger et al., ; goldenberg et al., ; houser et al., ; pallis et al., ) . differential diagnoses the differential diagnosis of peracute death in rabbits should include infection with clostridium spp. and/or ehec infection (garcia et al., ; perkins et al., ) . treatment, prevention, and control as with c. spiroforme, the reduction of risk factors and the prudent use of antibiotics are recommended (agnoletti et al., ) . cholestyramine may also be used for prevention (lipman et al., ) . fecal flora transplants have been suggested and commercial probiotic strains are able to inhibit c. difficile and c. perfringens in vitro (carman and evans, ; schoster et al., ) . research complications the sporadic nature of deaths due to c. difficile infection is unlikely to result in significant complications to research. iii. clostridium perfringens c. perfringens type e produces alpha and iota toxins and is an laboratory animal medicine uncommon cause of enterotoxemia in rabbits (redondo et al., ; songer, ) . because of the similarity between the iota toxins of c. spiroforme and c. perfringens type e, detection of toxin alone for diagnostic purposes will not differentiate between the two organisms (songer, ) . pcr can be used for typing c. perfringens based on amplification of toxin genes (daube et al., ) . historically, a disease process associated with e. coli infection was known as colibacillosis. currently, e. coli is classified based on the virulence factors that are genetically encoded and expressed in the bacteria. different virulence factors are associated with different e. coli 'pathotypes'. pathotypes may be associated with three general clinical syndromes: enteric/diarrheal disease, urinary tract infections, and sepsis/meningitis (kaper et al., ) . the centers for disease control and prevention currently recognizes six pathotypes associated with diarrhea in humans: enteropathogenic e. coli (epec), shiga toxin (stx)-producing e. coli (stec; also known as enterohemorrhagic e. coli (ehec) or verocytotoxin-producing e. coli (vtec)), enterotoxigenic e. coli (etec), enteroaggregative e. coli (eaec), enteroinvasive e. coli (eiec), and diffusely adherent e. coli (daec) (http://www.cdc.gov/ecoli/general/). comparative genomic analyses identified genes that were isolateand pathovar-specific and clustered strains according to pathotypes (lukjancenko et al., ; rasko et al., ) . two more emerging pathotypes have been suggested: adherent invasive e. coli (aiec; associated with crohn's disease in humans) and shiga toxin-producing enteroaggregative e. coli (steaec; associated with a large outbreak of hemolytic uremic syndrome (hus) in europe) (clements et al., ) . of these pathotypes, epec and stec are associated with natural disease in rabbits (cantey and blake, ; garcia et al., ) . in addition, necrotoxigenic e. coli (ntec) are associated with disease in rabbits (blanco et al., ) . pathogenic animal and human strains are very closely related and have virulence genes in common (clermont et al., ) . therefore, it is important to determine which e. coli pathotype(s) are associated with disease in rabbits in order to characterize new diseases and/or more accurately diagnose, prevent, control, and treat the condition as well as for epidemiological investigations. etiology epec carry the eae gene that encodes intimin, a protein involved in induction of attaching and effacing lesions in the intestine. e. coli serotype o , also known as rdec- , is the prototype epec strain which was isolated from rabbits with diarrhea and has been used experimentally as a model to study epec-induced disease (cantey and blake, ) . epec is an important cause of potentially fatal infant diarrhea in developing countries (kaper et al., ; swennes et al., ) . clinical signs ehec o was isolated from an outbreak of bloody diarrhea and sudden death in dutch belted rabbits (fig. . ) (garcia et al., ) . acute diarrhea following shipment was associated with epec o :h infection in laboratory rabbits (swennes et al., ) . laboratory rabbits can be reservoir hosts of pathogenic e. coli without exhibiting clinical signs (garcía and fox, ; swennes et al., ) . patent or occult blood may be detected in the feces of infected rabbits (camguilhem and milon, ; garcia et al., ) . epizootiology epec and ehec can be enzootic in rabbit colonies and these bacteria are transmitted by the fecal-oral route (garcía and fox, ; swennes et al., ; swennes et al., ) . epec and ehec coinfections are possible (garcía and fox, ; garcia et al., ) . ehec are a subset of stec that carry stx gene(s) that encode stx(s) and also carry the eae gene that encodes intimin (melton-celsa et al., ) . rabbits can harbor stec strains and are recognized as their vectors and reservoir hosts (bailey et al., ; blanco et al., ; garcía and fox, ; kim et al., ; leclercq and mahillon, ; pohl et al., ; pritchard et al., ; scaife et al., ) . pathology grossly, paintbrush hemorrhages of the cecal serosa may be observed after experimental infection with epec (camguilhem and milon, ) . also, experimentally, the serosal surface of the cecum and/or proximal colon can develop petechial or echymotic hemorrhages and may become edematous and thickened (garcía et al., ) . histologically and ultrastructurally, attaching and effacing lesions with pedestal formation can be observed with epec or ehec infections (kaper et al., ; peeters et al., ) . enterocolitis, nephropathy, and thrombotic microangiopathy can be observed in ehec-infected rabbits (garcía et al., ) . diagnosis feces or intestinal contents can be enriched in broth and then cultured using blood agar, macconkey agar, or ehec-selective media such as sorbitol macconkey agar or raibow® agar (garcía and fox, ; tarr, ; tarr et al., ) . after isolation of e. coli in pure culture, samples can be biotyped using commercial methods such as the api® e strips (bio-mérieux). serotyping and molecular characterization of isolates can be performed by the e. coli reference center (http://ecoli.cas.psu.edu/) at the pennsylvania state university. pcr assays can be utilized to detect virulence factors characteristic of epec, ehec, or other pathogenic e. coli as well as for high-resolution genotyping for epidemiological studies (blanco et al., ; garcía and fox, ) . molecular characterization of stec strains can be performed by the stec center (http://www. shigatox.net/new/) at michigan state university. differential diagnoses the differential diagnoses should include other causes of diarrhea in rabbits including the clostridial diseases and intestinal coccidiosis. treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown pathogenic e. coli status into a colony. rabbits should be screened by culture and e. coli isolates characterized for virulence factors by pcr. also, since it is known that ehec can contaminate plants and vegetables, laboratory personnel should be aware that rabbit feeds such as hay, alfalfa, and other greens have the potential to introduce enteric pathogens such as ehec into laboratory rabbits (berger et al., ; garcía and fox, ) . ehec o can survive for days in grass hay feed (davis et al., ) . cesarean section rederivation and antibiotic treatment have been suggested for eradication of pathogenic e. coli in rabbits (swennes et al., ) . a 'one health' approach should be incorporated to control ehec infections because outbreaks such as with ehec o in humans was linked to consumption of spinach contaminated by feral swine and was additionally isolated from domestic cattle, surface water, sediment, and soil (garcia et al., ) -a good example of integrating human, animal, and environmental health (monath et al., ) . antibiotic treatment should be based on culture and sensitivity. importantly, in humans infected with ehec, treatment with antibiotics is controversial due to the possibility of induction of stx-encoding bacteriophages and worsening of the clinical condition due to hemolytic uremic syndrome (tarr et al., ) ; therefore, antibiotic treatment of rabbits infected with ehec may not be recommended. clinically affected rabbits can be treated with fluids as this intervention is nephroprotective in humans (hickey et al., ) . in addition, rabbit epec strains may carry extended-spectrum beta-lactamases making them resistant to antibiotics (poeta et al., ) . parenteral enrofloxacin administered prior to shipment decreased morbidity and mortality associated with endemic epec (swennes et al., ) . research complications epec infection can cause high morbidity and mortality in laboratory rabbit colonies and can affect studies involving intestinal physiology in rabbits. epec and ehec present a zoonotic risk (garcia et al., ; poeta et al., ; swennes et al., ) . etiology treponema paraluiscuniculi is a noncultivable species that infects rabbits and causes venereal spirochetosis or treponematosis (also known as rabbit syphilis, vent disease, or cuniculosis) (smajs et al., ) . although its genome structure is closely related to other pathogenic treponema species including t. pallidum subsp. pallidum, the etiological agent of human syphilis, t. paraluiscuniculi does not infect humans (smajs et al., ) . genome sequencing revealed that t. paraluiscuniculi evolved from a t. pallidum-like ancestor and adapted to rabbits during loss of infectivity to humans (smajs et al., ) . t. paraluiscuniculi can also infect hares, and causes seroconversion, but no clinical signs. in contrast, the related organism, t. paraluisleporis, can infect and induce disease in rabbits and hares. the close phylogenetic association between t. paraluiscuniculi and t. paraluisleporis suggests that these organisms could be given a subspecies or ecovar status rather than species status (lumeij et al., ) . clinical signs in naturally infected rabbits lesions commonly occur in the vulva or prepuce (cunliffe-beamer and fox, a) . other parts of the body that may be affected, in descending order, include the anal region, nose, eyelid, and lip (cunliffe-beamer and fox, a) . naturally infected rabbits develop lesions of the ear, face, prepuce, and anus (small and newman, ) . in a study involving intratesticular inoculation of t. paraluiscuniculi, single lesions were found in the prepuce or scrotum and multiple lesions were found in the nose, mouth, ear, prepuce, foot, and scrotum (small and newman, ) . all lesions had abundant treponemes by dark-field examination (small and newman, ) . epizootiology susceptibility to, and expression of venereal spirochetosis, may vary with the strain of rabbit (cunliffe-beamer and fox, b) . the prevalence of t. paraluiscuniculi infection increased with parity in adult females and most adult males seroconverted within months of entering the breeding program. these findings suggested that t. paraluiscuniculi spreads by horizontal transmission in adult rabbits (digiacomo et al., ) . in an enzootically infected conventional rabbit colony, the frequency of venereal spirochetosis was lower in rabbits less than months of age than in adult rabbits (cunliffe-beamer and fox, b) . experiments involving cross fostering of newborns indicated that infection occurred at birth (vertical transmission) and during the suckling period (cunliffe-beamer and fox, b) . in addition, horizontal transmission by coitus and skin contact occurs (small and newman, ) . experimental topical or intradermal-subcutaneous genital inoculation of adult rabbits confirmed these routes of transmission (cunliffe-beamer and fox, b) . the t. pallidum repeat (tpr) genes in t. pallidum subsp. pallidum are thought to code for potential virulence factors. tprk was the only tpr homolog found in t. paraluiscuniculi that induced antibody and t cell responses after experimental inoculation of rabbits indicating that tprk may be an important virulence factor in venereal spirochetosis (giacani et al., ) . virulence factors and pathogenesis have been recently reviewed (smajs et al., ) . pathology the lesions include erythemathous macules or papules to erosions, ulcers, and crusts (cunliffe-beamer and fox, a) . diagnosis serologic tests that have been used include the nontreponemal antigen tests (venereal disease research laboratory (vdrl) and rapid plasma reagin), microhemagglutination, and fluorescent treponemal antibody absorption tests (digiacomo et al., ) . although the nontreponemal antigen tests were not completely satisfactory, the vdrl test was more sensitive and the plasma reagin test was more specific in detecting t. paraluiscuniculi infection (digiacomo et al., ) . the sensitivity and specificity of the microhemagglutination test compared favorably with the fluorescent treponemal antibody absorption test and was recommended as the optimal assay to make a diagnosis (digiacomo et al., ) . detection of t. paraluiscuniculi in lesions can be achieved by dark-field microscopic examination of scrapings from lesions and by histological evaluation of silver-stained testicular sections (cunliffe-beamer and fox, a; faine, ) . pcr has been used for molecular characterization of treponemes including t. paraluiscuniculi (cejkova et al., ) . differential diagnoses the skin lesions may be confused with abrasions (trauma), mycotic infections, and lesions of ectoparasites (acariasis) (small and newman, ) . treatment, prevention, and control there are no vaccines available at this time to prevent treponematosis in rabbits; however, rabbits have been used as experimental models to test vaccines against t. pallidum in humans (ho and lukehart, ) . hysterectomy derivation can eliminate venereal spirochetosis (cunliffe-beamer and fox, b) . a study investigating two different doses ( , or , iu/kg body weight/ week) of benzathine penicillin g-procaine penicillin g to treat rabbits at -day intervals found that both dosages were effective. lesions healed within weeks of the first treatment and the plasma reagin titers declined markedly or disappeared by the sixth week after the first treatment (cunliffe-beamer and fox, c) . research complications t. paraluiscuniculi can affect studies of t. pallidum in rabbits (small and newman, ) . partial immunological cross-protection has been observed between t. paraluiscuniculi and t. pallidum (smajs et al., ; turner and hollander, ) . etiology lawsonia intracellularis is a gram-negative, curved to spiral-shaped, obligate intracellular bacterium that causes proliferative enteropathy in rabbits and other species of animals (sait et al., ; schauer et al., ) . clinical signs an intraepithelial 'vibrio' was associated with acute typhlitis in rabbits - weeks after weaning (moon et al., ) . diarrhea was reported in japanese white rabbits with presumptive l. intracellularis infection and histiocytic enteritis (umemura et al., ) . in another report, sucklings and weanlings were affected and the feces of most of the rabbits were characterized as semifluid and mucinous or pasty, and three rabbits had watery diarrhea (schoeb and fox, ) . these affected rabbits were afebrile and lethargic, refused food and water, and most died within a few days after the onset of diarrhea (schoeb and fox, ) . diarrhea, depression, and dehydration that resolved over the course of - weeks were reported in -to -week-old new zealand white (nzw) rabbits (hotchkiss et al., ) . diarrhea and weight loss were reported in a -month-old rabbit (horiuchi et al., ). an outbreak of diarrhea with high ( %) mortality was reported in -to -month-old nzw rabbits with proliferative enterocolitis associated with l. intracellularis and epec (schauer et al., ) . epizootiology proliferative enteropathy generally occurs as isolated cases or occasional minor outbreaks in species other than the pig, blue fox, and hamster (lawson and gebhart, ) . infected rabbits can serve as reservoir hosts for l. intracellularis infection in other species including foals (pusterla et al., a (pusterla et al., , . however, l. intracellularis appears to adapt to the specific animal species it infects (vannucci et al., ) . the pathogenesis of l. intracellularis infection has been reviewed (lawson and gebhart, ; smith and lawson, ) . studies using interferon (ifn)-gamma receptor knockout mice determined that interferon ifngamma plays a significant role in limiting intracellular infection and increased cellular proliferation associated with l. intracellularis infection (smith et al., ) . lawsonia surface antigen (lsaa) plays a role during l. intracellularis attachment to and entry into intestinal epithelial cells (mccluskey et al., ) . balb/ca mice are susceptible to rabbit l. intracellularis isolates but not to pig l. intracellularis isolates suggesting that there are biological differences between the proliferative enteropathy isolates from rabbits and pigs (murakata et al., ) . pathology distention and diffuse mucosal thickening of the jejunum and proximal ileum with enlarged cranial mesenteric lymph nodes was observed in -to -month-old rabbits (umemura et al., ) . thickening of the mucosa was associated with distention of the lamina propria with macrophages and the enlargement of the lymph nodes was also associated with infiltration of macrophages (umemura et al., ) . minute bacilli were observed in the apical cytoplasm of mucosal epithelial cells using toluidine blue (umemura et al., ) . thickening of the cecum and proximal colon has also been reported (hotchkiss et al., ) . in another study, no gross lesions were found in the small intestine, but two suckling rabbits had reddened ceca with congested vessels (schoeb and fox, ) . in this study two types of microscopic lesions were characterized: ( ) erosive and suppurative cecitis and colitis, and ( ) proliferative lesions in the cecum, sacculated colon, ileum, and distal jejunum, or a combination of these (schoeb and fox, ) . some animals had both erosive and proliferative lesions. narrow curved or spiral bacteria were detected in rabbits with erosive and proliferative lesions using warthin-starry stain and these bacteria were more abundant in cases with severe lesions (schoeb and fox, ) . proliferative intestinal lesions contained curved to spiral argyrophilic intracellular bacteria in the apical cytoplasm of crypt enterocytes (hotchkiss et al., ) . diagnosis the s ribosomal dna sequences from l. intracellularis isolates from different species of animals are highly similar (cooper et al., a) . however, antigenic differences have been found between pig and rabbit isolates . the complete genome sequence of a porcine strain has been recently reported (sait et al., ) . l. intracellularis can be detected in feces from healthy and diarrheic rabbits (lim et al., ) . pcr assays to detect l. intracellularis dna in feces have been evaluated (pedersen et al., ) . these assays can be used for ante mortem diagnosis of proliferative enteropathy in pigs (pedersen et al., ) . pcr primers used to diagnose lawsonia in other animals species have been used in rabbit cases (cooper et al., b; duhamel et al., ; fox et al., ; horiuchi et al., ; hotchkiss et al., ; jones et al., ) . other diagnostic methods include enzyme-linked immunosorbent assay (elisa) using synthetic peptides of lsaa and immunomagnetic separation using anti-lsaa antibody to capture l. intracellularis in fecal samples followed by detection with atp bioluminescence (watarai et al., (watarai et al., , . in tissue sections, l. intracellularis can be detected using silver stains such as warthin-starry stain (duhamel et al., ; horiuchi et al., ; hotchkiss et al., ; schauer et al., ; schoeb and fox, ) . indirect immunofluorescence has also been used in deparaffinized intestinal sections from infected rabbits (schoeb and fox, ) . immunohistochemistry using antiserum against synthetic peptides of lsaa has also been used to detect l. intracellularis in the ileum of a naturally infected rabbit (watarai et al., ) . electron microscopy reveals organisms that are ~ . - . μm wide and ≤ . μm long in the apical cytoplasm of villous and crypt epithelial cells (duhamel et al., ) . l. intracellularis can be cultured from homogenized intestinal tissue in cell lines including iec- (rat small intestinal cells) and mccoy cells (mouse fibroblasts) (lawson and gebhart, ; watarai et al., ) . a quantitative pcr (qpcr) assay that is able to assess the growth of l. intracellularis in cultured cells has also been used to detect the organisms in pig fecal samples and could be used in other animal species (drozd et al., ) . differential diagnoses clinically, the differential diagnosis should include other causes of diarrhea in rabbits. mycobacterium avium subsp. paratuberculosis can infect rabbits and induce thickening of the intestinal mucosa (beard et al., ; greig et al., ) . therefore, rabbit intestinal sections should be examined for acidfast organisms using stains such as ziehl-neelsen stain (duhamel et al., ; horiuchi et al., ; schoeb and fox, ; umemura et al., ) . furthermore, other intestinal organisms may colonize the intestine during l. intracellularis infection in rabbits (duhamel et al., ; hotchkiss et al., ; lim et al., ; schauer et al., ) . other bacterial diseases have been sporadically reported in rabbits. these are summarized in table . . treatment, prevention, and control vaccination strategies have been tested and developed for pigs and horses, but not for rabbits (nogueira et al., ; pusterla et al., b; weibel et al., ) . testing rabbits by pcr prior to introduction to a laboratory colony may be necessary for exclusion of this organism. oral neomycin ( mg/rabbit) was used to treat surviving rabbits during an outbreak of presumptive l. intracellularis and the diarrhea subsided (umemura et al., ) . because l. intracellularis infects the intestine and ifn-gamma appears to be involved in pathogenesis, research involving rabbit gastrointestinal pathology and immune responses may be confounded by infection with this organism. research complications the mortality associated with l. intracellularis infection would be disruptive to ongoing studies. etiology myxomatosis is caused by myxoma virus, a member of the family poxviridae, genus leporipoxvirus (kerr and donnelly, ; spiesschaert et al., ) . clinical signs the severity of disease varies with the strain of virus and the host species and breed. rabbits of the genus oryctolagus are particularly susceptible and often develop a fatal disease characterized by mucinous skin lesions and tumors. affected animals also exhibit edema around the mouth, nose, anus, and genitals as well as progressive conjunctivitis with serous and mucopurulent secretions from the eyes and nose (brabb and di giacomo, ; kerr and donnelly, ; spiesschaert et al., ) . bacterial pneumonia commonly develops and animals die - days after infection. the virus is spread by arthropod vectors and direct contact. epizootiology myxomatosis has a worldwide distribution. various species of sylvilagus and lepus are naturally susceptible (brabb and di giacomo, ) . the myxoma virus genome encodes for a number of immunomodulatory proteins which greatly affect the host immune response by inhibiting apoptosis, interfering with leukocyte chemotaxis, and suppressing leukocyte activation, thereby fostering viral replication and spread (spiesschaert et al., ) . pathology histopathology shows these 'myxomas' to be composed of undifferentiated stellate mesenchymal cells embedded in a matrix of mucinous material and interspersed with capillaries and inflammatory cells (brabb and di giacomo, ; kerr and donnelly, ) . diagnosis diagnosis can be made by pcr or elisa. definitive diagnosis depends on culture of the virus from infected tissues. differential diagnoses rabbits of the genus sylvilagus develop fibroma-like lesions that may be indistinguishable from those caused by rabbit fibroma virus. the two diseases have been distinguished by inoculation of fibroma material into oryctolagus rabbits. they develop fatal disease if the myxoma virus is the etiologic agent, or fibromas if rabbit fibroma virus is responsible. treatment, prevention, and control since the disease is spread by fleas and mosquitoes as well as by direct contact, control measures should include prevention of contact with arthropods and quarantine of infected rabbits. vaccines have been used in europe with some success. most recently, a live recombinant vaccine for both myxomatosis and rabbit hemorrhagic disease has been released in the united kingdom (spibey et al., ) . research complications none have been reported. rabbit (shope) fibroma virus is a leporipoxvirus that is antigenically related to myxoma virus. fibromatosis is endemic in wild rabbits; however, an outbreak in commercial rabbits caused extensive mortality (joiner et al., ) . usually, less virulent strains cause skin tumors in domestic rabbits (raflo et al., ) . the disease is probably spread by arthropods (brabb and di giacomo, ; kerr and donnelly, ) . fibromas are flat, subcutaneous, easily movable tumors, and most often found on the legs and face. tumors may persist for some time but eventually regress. metastasis does not occur. rabbit pox is a rare disease induced by an orthopoxvirus taxonomically similar to vaccina virus that has caused outbreaks of fatal disease in laboratory rabbits in the united states and holland (brabb and di giacomo, ) . rabbits with the disease may or may not present with 'pox' lesions in the skin. the animals have a fever and nasal discharge or days after infection. most rabbits have eye lesions including blepharitis, conjunctivitis, and keratitis with subsequent corneal ulceration. skin lesions, when present, are widespread. they begin as a macular rash and progress to papules up to cm in diameter by days postinfection. the lymph nodes are enlarged, the face is often edematous, and there may be lesions in the oral and nasal cavity. at gross necropsy, nodules can be found in the liver, gall bladder, spleen, lung, and reproductive organs. necrosis is widespread. characteristic cytoplasmic inclusions seen in many poxvirus infections are rare in this disease. mortality is high in affected animals. the virus is apparently spread by aerosols and is difficult to control. rabbit pox is used as a model of smallpox in humans in response to the potential use of smallpox as a bioterrorism agent. it is an effective model for the evaluation of potential therapies against smallpox (nalca and nichols, ; rice et al., ) . four herpesviruses (leporid herpesviruses , , , and ) have been isolated from rabbits and hares (davison, ) . leporid herpesvirus (lhv- ) was isolated from cottontail rabbits and is also known as cottontail rabbit herpesvirus. it is not pathogenic for domestic rabbits. lhv- (herpesvirus cuniculi) was isolated from domestic rabbits (o. cuniculus) and causes subclinical infections. lhv- (herpesvirus sylvilagus) was isolated from cottontail rabbits. cottontail rabbits infected with the virus develop a lymphoproliferative disease with lymphoid infiltration of many organs (hesselton et al., ) . lhv- does not infect domestic rabbits. lhv- - are tentatively classified in the genus radinovirus, subfamily gammaherpesvirinae. lhv- was isolated from domestic rabbits and caused severe disease in preweanlings (jin et al., a, b) . clinical signs included weakness, anorexia, conjunctivitis, keratitis, and periocular swelling. some animals also developed skin ulcers. lhv- is genus simplexvirus, subfamily alphaherpesvirinae. the cottontail rabbit is the natural host of the cottontail (shope) papillomavirus, a kappapapillomavirus, laboratory animal medicine which causes horny warts primarily on the neck, shoulders, and abdomen. the disease has a wide geographic distribution with the highest incidence occurring in rabbits in the midwest (brabb and di giacomo, ) . as many as % of infected sylvilagus rabbits develop squamous cell carcinomas. natural outbreaks in domestic rabbits have been reported (hagen, ) . in these natural outbreaks, papillomas were more common on the eyelids and ears. the virus is transmitted by arthropod vectors. this virus is used extensively as a model for the study of oncogenic virus biology and as a model for the treatment and prevention of papillomavirus infections in humans (christensen, ; salmon et al., ; sundarum et al., ) . oral papillomatosis in domestic rabbits is caused by a kappapapillomavirus that is related to but distinct from the cottontail rabbit papilloma virus. naturally occurring lesions have been seen in laboratory rabbits and appear as small, white, discrete growths on the ventral surface of the tongue (kerr and donnelly, ) . lesions may ulcerate. microscopic examination shows them to be typical papillomas. most lesions eventually regress spontaneously (brabb and di giacomo, ; kerr and donnelly, ) . etiology rabbit rotavirus is a member of the family reoviridae. all isolates of rabbit rotavirus have been classified as group a and have been serotype (brabb and di giacomo, ; kerr and donnelly, ) . clinical signs the severity of disease in naturally occurring outbreaks has been variable. in severe outbreaks, affected animals exhibit anorexia, dehydration, and watery to mucoid diarrhea and mortality can be quite high. in other reported outbreaks, mild, transient diarrhea has been reported (brabb and di giacomo, ; kerr and donnelly, ) . similarly, attempts to experimentally produce clinical disease have had variable results. mild diarrhea is usually seen, but in some studies there has been significant mortality. it is probable that other factors, such as maternal antibodies, diet, and the presence of pathogenic bacteria, affect the severity of clinical disease in outbreaks. for example, in combined experimental infections with both rotavirus and e. coli, the inoculation of both organisms led to more serious clinical signs than when given alone, indicating that rotavirus may have been a more significant determinant in the manifestation of this disease (thouless et al., ) . these investigators also showed that older rabbits were naturally more resistant to the combined infection with rotavirus and e. coli. epizootiology rotavirus infections of domestic rabbits are common (brabb and di giacomo, ) . many colonies of rabbits are serologically positive, and rotavirus can be isolated readily from rabbit feces. in endemically infected colonies, maternal antibodies to rotaviruses are passed transplacentally and decline at around the time of weaning (brabb and di giacomo, ) . rabbits of weaning age are most susceptible. very young rabbits appear to be protected from rotavirus infection by passive immunity, when present, but are quite susceptible when there is none (schoeb et al., ) . this is also the time when they are most likely to be subjected to diet changes that may contribute to a change in microbial flora. pathology in affected animals, there is villous atrophy and loss of epithelial cells in the small intestines. a lymphocytic infiltrate is present. diagnosis immunoassays (elisa and multiplex fluorescent immunoassay) are commercially available for rabbit rotavirus. a commercial immunochromatography kits for detecting human rotavirus infection was used successfully to diagnose rabbit rotavirus infection (fushuku and fukuda, ) . differential diagnoses c. piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, coccidiosis, and intestinal parasites should be considered. research complications colony mortality would be disruptive to ongoing studies. pleural effusion disease/infectious cardiomyopathy was diagnosed in rabbits inoculated with t. palliduminfected stocks of testicular tissue. because these treponemes could not be grown in vitro, the organism was propagated by passage in rabbits. the stocks were contaminated with a coronavirus, although it is not known whether this virus originated from rabbits or was a virus of human origin that had adapted to rabbits. with continued passage, the virus became more virulent, and significant mortality ensued. evidence indicated that it was not transmitted by direct contact. rabbits died due to congestive heart failure, and microscopic examination showed there was widespread necrosis of the heart muscle. it has been suggested that infection with this virus might be a model for the study of virus-induced cardiomyopathy (brabb and di giacomo, ; kerr and donnelly, ) . rabbit enteric coronavirus has been isolated from tissue cultures from rabbits (brabb and di giacomo, ; kerr and donnelly, ; lapierre et al., ) and has been associated with one naturally occurring outbreak of diarrhea in a barrier-maintained breeding colony (eaton, ) . these rabbits developed severe diarrhea, and most died within h of onset of clinical signs. attempts to reproduce the disease led to watery diarrhea, which lasted a short time; however, none of the rabbits died. it is quite probable that other microorganisms or laboratory animal medicine unknown environmental factors contributed to the severity of this outbreak. etiology rabbit hemorrhagic disease virus is a calicivirus of the genus lagovirus and is the causative agent of rabbit hemorrhagic disease (rhd) (abrantes et al., ; brabb and di giacomo, ; kerr and donnelly, ) . clinical signs three clinical syndromes are seen (abrantes et al., ) . the peracute form is characterized by sudden death without clinical signs. acutely affected animals demonstrate anorexia and depression. in addition, neurologic signs, respiratory signs, ocular hemorrhage, and epistaxis may be seen. morbidity and mortality are extremely high. lymphopenia and abnormalities in coagulation parameters are also seen. in the subacute form, similar signs may occur but are considerably milder and most of these rabbits survive (abrantes et al., ; kerr and donnelly, ) . epizootiology rabbit hemorrhagic disease was first reported in china in and is currently endemic in europe, asia, africa, australia, and new zealand. in addition, isolated outbreaks have been reported in numerous countries. the virus is transmitted by the fecal-oral route. the role of fomites and arthropod vectors is also suspected (brabb and di giacomo, ; kerr and donnelly, ) . the incubation period may be as short as or days, and sudden death with no previous signs is common. pathology periportal hepatic necrosis is the only consistent microscopic lesion, and the animals die due to disseminated intravascular coagulopathy and thrombosis (abrantes et al., ; kerr and donnelly, ) . diagnosis the virus has not been successfully grown in vitro; however, diagnosis can be confirmed with negative-contrast electron microscopy of liver tissue. specific antibodies can be detected by elisa or by hemagglutination inhibition. differential diagnoses a related calicivirus, european brown hare virus, has caused disease in hares in several countries in europe (brabb and di giacomo, ) . animals present with necrotic hepatitis, hemorrhages in the trachea and lungs, and pulmonary edema. a monoclonal antibody elisa is available for serodiagnosis, and control measures are similar to those for rhd. the agent resists drying, can be carried on fomites, and may be transmitted via respiratory and intestinal secretions (mitro and krauss, ) . any rabbit colonies with this disease should be quarantined and depopulated, and the environment thoroughly cleansed and disinfected. research complications colony mortality would be disruptive to ongoing studies. other viral infections several other viruses have been isolated from rabbit tissues, but have not been shown to produce disease. these include paramyxoviruses and bunyaviruses. serologic titers to togaviruses and flaviviruses have also been demonstrated in rabbits (brabb and di giacomo, ; kerr and donnelly, ) . etiology hepatic coccidiosis is caused by the parasite eimeria stiedae, which has also been referred to as monocystis stiedae, coccidium oviforme, and c. cuniculi (hofing and kraus, ) . clinical signs the clinical disease has a wide range of manifestations. mild infections often result in no apparent disease. most clinical signs are the result of interruption of normal hepatic function and blockage of the bile ducts. these signs are more common in juvenile rabbits and can include hepatomegaly, icterus, and anorexia (schoeb et al., ) . diarrhea can occur at the terminal stages of the disease (hofing and kraus, ) . decreased growth rates and weight loss are common. joyner et al. ( ) demonstrated that infected rabbits begin to lose weight within days. enlargement of the liver (hepatomegaly) is common. the liver normally is approximately . % of the body weight, but rabbits with severe hepatic coccidiosis may have livers that contribute to greater than % of the body weight (lund, b) . the age of the host strongly affects parasite development and oocyst production. four-month-old, coccidia-free rabbits experimentally infected with e. stiedae produced fewer oocysts than similarly infected -monthold rabbits (gomez-bautista et al., ) . epizootiology e. stiedae is found worldwide, although rabbits bred for use in research are commonly free of the parasite. transmission occurs by the fecaloral route, as for other coccidia. the organism has also been experimentally transmitted by intravenous, intraperitoneal, and intramuscular administration of oocysts (pellérdy, ) . smetana ( ) demonstrated that infection of the entire liver occurred following ligation of the right bile duct and inoculation of e. stiedae oocysts. the study also showed that infection occurred earliest within the small intrahepatic ducts, leading to the theory that infection occurred via blood or lymph. the precise life cycle is still undetermined, although a number of studies have examined it (horton, ; owen, ; rose, ) . sporozoites have been demonstrated in the lymph nodes following experimental inoculation (horton, ; rose, ) . pathology necropsy often shows the liver to be enlarged and discolored, with multifocal yellowish laboratory animal medicine white lesions of varying size (fig. . ) . exudate in the biliary tree is common, along with dilatation of bile ducts. microscopically, papillomatous hyperplasia of the ducts along with multiple life-cycle stages of the organism can be observed in the biliary epithelium ( fig. . ) . diagnosis infected rabbits may have decreased fibrinogen when compared to uninfected rabbits (cam et al., ) . serum bilirubin levels can rise to mg/ dl, increasing as soon as day of infection and increasing through days - before moderating (rose, ) . leukocytosis and anemia can be observed and acute phase proteins are notably increased by days post infection (freitas et al., ) . diagnosis can be made by examination of fecal material, by either flotation or concentration methods. oocysts can also be detected within the gallbladder exudate (hofing and kraus, ) . alternatively, oocysts can sometimes be observed by microscopic examination of impression smears of the cut surface of the liver. ultrasonography may be a useful tool for diagnosis, with dilated vessels and bile ducts and increased echogenicity of the liver parenchyma (cam et al., ) . differential diagnoses the hyperplastic biliary ducts can be mistaken grossly for neoplasia. other types of parasitic hepatitis should be considered as differential diagnoses. less frequently, hepatitis secondary to bacterial infections can occur. treatment, prevention, and control control of the infection until development of natural immunity is one strategy to minimize the severity of disease. davies et al. ( ) demonstrated that immunity occurs following a light infection with e. stiedae. in the rabbit, immunity to eimeria may be lifelong (niilo, ; pellérdy, ) . prevention of hepatic coccidiosis with sulfaquinozaline in the feed ( ppm) was shown to prevent infection when experimental challenged with , sporulated oocysts (joyner et al., ) . sulfonamides have been shown effective against eimeria spp. (hagen, ; horton-smith, ; jankiewicz, ; lund, a; tsunoda et al., ) . treatment with toltrazuril ( ppm in drinking water for one day) has been shown to effectively treat infected animals (cam et al., ) . thorough sanitation of potentially contaminated surfaces is critical to control of coccidiosis. research complications potential research complications arising from hepatic coccidiosis are considerable. the resulting liver damage and decreased weight gains can complicate both the supply of rabbits for research as well as adversely affect research protocols. etiology there are at least different pathogenic species of intestinal coccidia in rabbits, including eimeria coecicola, e. elongate, e exigua, e. intestinalis, e. flavescens, e. irresidua, e. magna, e. matsubayashii, e. media, e. nagnurensis, e. neoleporis, e. piriformis, e. vejdovskyi, and e. perforans (pakandl, ). all of these coccidia are presented here as a group rather than as individual species of intestinal coccidia. clinical signs although intestinal coccidiosis may be subclinical, clinical signs can range from mild to severe and can result in death of the animal. postweanling rabbits are the most likely to experience mortality related to intestinal coccidiosis. suckling rabbits (< days old) are generally considered to be resistant to infection (pakandl and hlaskova, ) . clinical signs also depend on the species of coccidia that are present. severe diarrhea, weight loss, or mild reduction in growth rate are all laboratory animal medicine possibilities. fecal occult blood may be detected with e. perforans infection (li and ooi, ) . death is usually associated with severe dehydration subsequent to diarrhea (frenkel, ) . epizootiology intestinal coccidiosis is a common rabbit disease worldwide (varga, ) . transmission is by the fecal-oral route through ingestion of sporocysts. unsporulated oocysts are passed in the feces and are not infective. such oocysts will, however, sporulate to an infective stage within days after shedding; thus, it is important that sanitation be frequent enough to remove infective stages from the environment. the oocyst burden of feces can be enormous. gallazzi (gallazzi, ) demonstrated that a subclinical carrier of intestinal coccidia had , oocysts/gram of feces and that a rabbit with diarrhea could shed in excess of , oocysts/gram of feces. environmental contamination with oocysts can be a problem when large numbers of oocysts are being excreted. the life cycles of eimeria spp. are similar to those of other coccidia. schizogony, gametogony, and sporogony are the three phases of this life cycle. other sources can be consulted for greater detail on the life cycle of these protozoans (davies et al., ; pakandl, ; pakandl and jelinkova, ; pellérdy, ; rutherford, ) . pathology lesions are apparent in the small and large intestines. necrotic areas of the intestinal wall appear as white foci (pakes, ; pakes and gerrity, ) . the location and extent of the lesions depend on the species of coccidia. diagnosis diagnosis of intestinal coccidiosis can be made through identification of the oocysts in the feces (pakes and gerrity, ) . a pcr has been developed (oliveira et al., ) that differentiates between of the different eimeria species that infect the domestic rabbit. this test has excellent sensitivity, with the ability to detect . - . sporulated oocysts per sample. smaller scale pcr for detection and differentiation between the more pathogenic species (e. intestinalis, e. flavicenens, and e. stiedae) has also been developed (yan et al., ) . differential diagnoses other causes of diarrhea in rabbits should be considered including tyzzer's disease, the clostridial diseases, colibacillosis, l. intracellularis, enteric coronovairus and rotavirus, protozoons, or intestinal parasites. treatment, prevention, and control because intestinal coccidiosis is most common in postweanling rabbits, prevention of the disease should focus on the preweaning period. an oral vaccination has been developed and consists of a nonpathogenic strain of e. magna. this vaccine is sprayed into the nest box when rabbits are days of age. the preweanling rabbits develop immunity subsequent to infection with the nonpathogenic strain and are then resistant to wild-type strains of e. magna at days of age (drouet-viard et al., ) . other oral vaccines developed from various eimeria strains are also in development (akpo et al., ) . prevention and control of infection can be accomplished by providing . % sulfamerazine or . % sulfaquinoxaline in the drinking water (kraus et al., ) . a combination of sulfaquinoxaline, strict sanitation, and elimination of infected animals has been shown to eliminate intestinal coccidiosis from a rabbit breeding colony (pakes and gerrity, ) . as for hepatic coccidiosis, sulfaquinoxaline provided in the feed ( ppm) is an effective treatment. research complications intestinal coccidiosis can impact studies of the gastrointestinal tract, or have an impact on survival of postweanling rabbits. etiology the protozoan organism cryptosporidium cuniculus has been found in the intestinal tract of the rabbit (hadfield and chalmers, ; inman and takeuchi, ; kaupke et al., ; rehg et al., ; robinson et al., ; shiibashi et al., ; zhang et al., ) . clinical signs clinical signs related to cryptosporidiosis seem to be quite variable in the rabbit. a large farm outbreak (kaupke et al., ) had rabbits that presented with lethargy, anorexia and diarrhea. animals showing clinical signs died within - days. the stress of weaning is thought to have exacerbated these signs. another report describes small intestinal dilatation observed during surgery in a rabbit without other clinical signs (inman and takeuchi, ) . epizootiology transmission is likely via ingestion of thick-walled sporulated oocysts. experimentally infected juvenile rabbits began shedding oocysts in their feces - days post infection and continued to shed until days post infection without clinical signs (robinson et al., ) . pathology histopathology of the small intestine of the reported rabbit was characterized by shortened, blunted villi and mild edema of the lamina propria. the lacteals of the ileum were also dilated, and an inflammatory response was observed (inman and takeuchi, ) . diagnosis c. cuniculus is emerging as a potential zoonotic pathogen with several reports in recent years (chalmers et al., (chalmers et al., , zhang et al., ) . in response to this, real-time pcr assays are in development (hadfield and chalmers, ) that detect and differentiate c. cuniculus from c. parvum and c. hominis. differential diagnoses c. cuniculus can only be differentiated from c. hominis and c. parvum via genetic analysis (robinson et al., ) . differential diagnoses would include infection with clostridium piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, rotavirus, protozoans, or intestinal parasites. treatment, prevention, and control minimizing stress can possibly prevent or reduce clinical signs laboratory animal medicine (kaupke et al., ) . antibiotics were ineffective in the large farm outbreak. presumably, supportive care (fluids) would be indicated in animals showing clinical signs (schoeb et al., ) . prevention requires husbandry and sanitation practices that prevent exposure. research complications this organism is emerging as a human pathogen, so appropriate precautions should be made to protect research personnel from rabbits positive for c. cuniculus. etiology the etiologic agent responsible for encephalitozoonosis is encephalitozoon cuniculi. this agent is historically known by the name nosema cuniculi (pakes and gerrity, ) and has been divided into three strains (i -rabbit strain, ii -mouse strain, iiidog strain) (didier et al., ) . the disease was first described in as an infectious encephalomyelitis causing motor paralysis in young rabbits (wright and craighead, ) . clinical signs encephalitozoonosis typically has a delayed onset (weeks to months post infection) prior to the exhibition of clinical signs. early infection affects the kidney, liver and lung, while alterations later in the infection are most severe in the kidneys and brain (kunzel and joachim, ) . the organism can be found in the tissues without an inflammatory response (pakes and gerrity, ) . although named for the motor paralysis in young rabbits, the disease is usually latent. if clinical signs are present, they can include convulsions, tremors, torticollis, paresis, and coma (pattison et al., ) as well as signs of kidney failure. intrauterine infection can result in phacoclastic uveitis leading to rupture of the lens capsule (kunzel and joachim, ) . epizootiology transmission is likely horizontal via direct contact or environmental contamination (kunzel and joachim, ) , primarily from ingestion of infected urine (schoeb et al., ; wasson and peper, ) . the pathogen can also be transmitted vertically, as evidenced by in utero pcr positivity reported by baneux and pognan ( ) . pathology the kidneys commonly have lesions at necropsy. typically, there are multiple white, pinpoint areas or gray, indented areas on the renal cortical surface (kraus et al., ) . microscopically, these areas are characterized by granulomatous inflammation. interstitial infiltration of lymphocytes and plasma cells and tubular degeneration may also be present (flatt and jackson, ) . granulomatous encephalitis is a characteristic lesion (fig. . ) (pakes and gerrity, ) . lesions of the spinal cord can also occur (koller, ) . the organisms are often not observed in histologic sections of the lesions. organisms may be seen floating free in the tubules of the kidney (pakes and gerrity, ) . diagnosis diagnosis of encephalitozoonosis can be made using several different methods. histologic examination of tissues and observation of the organism is definitive. brain and kidney samples yield the best detection rates for histopathological diagnosis (leipig et al., ) . the encephalitozoon organism does not stain well with hematoxylin and eosin, and is better demonstrated using giemsa stain, gram stain, or goodpasture's carbol fuchsin stain (pakes, ) . many different serologic tests exist for the organism. indirect fluorescence antibody technique and elisa are both available and reliable (kunzel and joachim, ) . advances in diagnostic techniques have been made in human medicine due to the susceptibility of immunosuppressed patients to this particular infection. several pcr tests for diagnosis and species differentiation of encephalitozoonosis have been developed (croppo et al., ; franzen et al., ; weiss and vossbrinck, ) . pcr can be performed on the intestine, brain, heart, liver, lung, or kidney tissue with a good ( %) overall detection rate reported (leipig et al., ) . differential diagnoses if the animals are demonstrating motor paralysis, conditions such as splay leg should be considered. for neurological signs, consider bacterial meningitis due to p. multocida infection or rabbit hemorrhagic disease. treatment, prevention, and control prevention and control of the organism in the colony are done by elimination of the organism from the colony of infected rabbits. because this is a latent disease in rabbits, serologic methods must be used to identify carriers of the organism. the indirect fluorescence antibody test has laboratory animal medicine been used successfully to identify infected rabbits (cox, ) . the elimination of infected rabbits must be accompanied by disinfection of the environment. several disinfectants have been effective against this organism. encephalitozoon was killed by % (v/v) lysol, % (v/v) formalin, and % (v/v) ethanol (shadduck and polley, ) % hydrogen peroxide, and % sodium hydroxide (kunzel and joachim, ) . successful treatment and prevention of e. cuniculi in the rabbit has been reported with use of fenbendazole (suter et al., ) . for cases of phacoclastic uveitis, removal of the lens is the treatment of choice (kunzel and joachim, ) . research complications encephalitozoonosis is most commonly subclinical disease, which makes it difficult to determine the effects it may have on research. granulomatous reactions would complicate renal physiology and neurologic research. depression of the igg response and an increase in the igm response to brucella abortus antigens has been demonstrated in rabbits infected with encephalitozoon organisms (cox, ) . encephalitozoonosis is also a recognized disease in immunodeficient humans. it is recommended that such individuals seek medical counsel prior to handling rabbits. isolates from humans have been shown to be infectious for rabbits (mathis et al., ) . etiology psoroptes cuniculi is a nonburrowing mite and the causative agent of psoroptic mange, also called ear mange, ear canker, or otoacariasis. the organism is distributed worldwide, but with modern husbandry practices, it is mostly historical in laboratory rabbit colonies (schoeb et al., ) . clinical signs lesions occur primarily in the inner surfaces of the external ear. the lesions are pruritic and can result in scratching, head shaking, pain, and even self-mutilation (hofing and kraus, ) . a tan, crusty exudate accumulates in the ears over the lesions and can become quite extensive and thick (fig. . ) . the skin under the crust is moist and reddened. the ears may become malodorous. epizootiology all stages of the mite (egg, larva, protonymph, and adult) occur on the host. early in the infestation, mites feed on sloughed skin cells and lipids. as local inflammation increases, they ingest serum, hemoglobin, and red blood cells (deloach and wright, ; hofing and kraus, ) . the entire life cycle is complete in days. mites are relatively resistant to drying and temperature and can survive off the host for - days in a temperature range of - °c and relative humidity of - %. pathology lesions are characterized histologically by chronic inflammation, hypertrophy of the malpighian layer, parakeratosis, and epithelial sloughing. a hypersensitivity response to the mites, mite feces, and saliva likely contributes to lesions (hofing and kraus, ) . diagnosis mites are large enough to be seen with the unaided eye or with an otoscope. material scraped from the inner surface of the ear can also be examined using a dissecting microscope. mites are oval-shaped with well-developed legs that project beyond the body margin. adult males measure - μm × - μm, and females measure - μm × - μm (hofing and kraus, ) . differential diagnoses rarely, infection with sarcoptes scabiei or cheyletiella parasitovorax should be considered as differential diagnoses. treatment, prevention, and control several successful treatments have been reported. prior to local treatment, the ears should be cleaned gently to remove accumulated exudate. one treatment involves the application of % rotenone in mineral oil ( : ) every days for days. ivermectin is an effective treatment at dosages of - μg/kg sc or im (curtis et al., ; mckellar et al., ; wright and riner, ) . one or two doses were utilized for effective treatment. treatment of moderate to severe infestations with ivermectin alone can fail. using adjunct vitamin therapy to minimize oxidative tissue damage has been shown to enhance treatment success (singh et al., ) . a single dose of topical selamectin at a minimum of mg/kg selamectin (kurtdede et al., ) and a single injection of laboratory animal medicine eprinomectin at or μg/kg (pan et al., ) were found to be effective treatments. regardless of treatment modality, it is generally recommended that the entire group of rabbits be treated at the same time. heat ( °c) and desiccation (< % humidity) will kill parasites that are not on the host (arlain et al., ) . vaccine targets have been investigated, with gut surface antigen being the primary focus (rossi et al., ) . research complications p. cuniculi has been associated with immune suppression and a systemic inflammatory reaction (shang et al., ) . ear trauma secondary to psoroptes infestation can limit access to the auricular artery and veins. . cheyletiella spp. (c. parasitovorax, c. takahasii, c. ochotonae, c. johnsoni) etiology cheyletiella mites are nonburrowing skin mites of rabbits. they are distributed worldwide. several closely related species have been reported to occur on rabbits, namely, c. parasitovorax, c. takahasii, c. ochotonae, and c. johnsoni (hofing and kraus, ) . clinical signs the anatomic site most commonly infested is the area over the scapulae. there may be mild hair loss in the area, and the skin may have a gray-white scale (cloyd and moorhead, ) . affected rabbits do not scratch, and there is no evidence of pruritus. skin lesions are mild or nonexistent. epizootiology all stages (egg, larva, pupa, and adult) in the life cycle occur on the host. mites remain in association with the keratin layer of the skin and feed on tissue fluid (myktowycz, ) . transmission is probably by direct contact (schoeb et al., ) . pathology when present, skin lesions are characterized by mild dermatitis, hyperkeratosis, and an inflammatory cell infiltrate (hofing and kraus, ) . diagnosis mites can be isolated by scraping or brushing fur in the affected areas onto a slide. clearing samples with - % potassium hydroxide will improve visibility of the mites, which can then be identified using a dissecting microscope. the female measures × μm, and the male is × μm. cheyletiella mites have a large, distinctive curved claw on the palpi (pegg, ) . differential diagnoses other skin mites (such as sarcoptes scabei) or fur mites (leporacarus gibbus) that can affect rabbits should be considered as well as the possibility of dermatophytosis. treatment, prevention, and control topical acaricides are often used and are effective at controlling infestation. ivermectin (subcutaneous or subcutaneous and oral) and selamectin (topical) treatments have been used successfully. eggs in the environment can reinfest the host, so posttreatment environmental sanitation is important (mellgren and bergvall, ) . research complications cheyletid mites can cause a transient dermatitis in humans who are in close contact with infested animals (cohen, ; lee, ) . for this reason, these mites can be considered a zoonotic pathogen. etiology sarcoptes scabiei is a burrowing mite and the causative agent of sarcoptic mange. mites of the genus sarcoptes are generally considered to be one species, s. scabiei, but are often further identified by a variety name corresponding to the host species (e.g., s. scabiei var. cuniculi). the organisms are commonly referred to as itch or scab mites. the disease has a worldwide distribution. clinical signs affected rabbits will exhibit intense pruritus with hair loss and abrasions as a resulting from scratching. serous encrustations on the skin and secondary bacterial infections are common. there has been one report of a secondary infection with the yeast malassezia (radi, ) . anemia and leukopenia can also be observed in affected rabbits (arlain et al., ) . epizootiology sarcoptic are similar to notoedric mites (notoedres cati) in morphology, life cycle, and public health significance. mites burrow and produce an intensely pruritic dermatitis. lesions are most common on the head (hofing and kraus, ) . all stages of sarcoptic mange mites occur on the host. the females burrow into the skin to lay eggs. young larvae can also be found in the skin, whereas older larvae, nymphs, and males reside on the skin surface. mites feed on lymph and epithelial cells (hofing and kraus, ) . pathology amyloidosis of the liver and glomerulus have been reported in rabbits with severe infestation (arlain et al., ) . the skin itself is hyperplastic and hyperkeratotic, with inflammatory response evident in the dermis (schoeb et al., ) . diagnosis because sarcoptes is a burrowing mite, skin scrapings are necessary to diagnose infestation. samples may be cleared with - % potassium hydroxide. female mites measure - μm × - μm. the body shape is round, and the legs are very short. differential diagnoses other causes of dermatitis in rabbits (such as cheyletiella spp., p. cuniculi or dermatophytosis) should be considered. ivermectin is effective at eliminating infestation at μg/kg administered subcutaneously. a single topical dose of selamectin at - mg/kg reduced the number of mites found on skin scrapings of angora rabbits (kurtdede et al., ) and eliminated clinical signs and parasitic infestation in a group of mixed-breed rabbits at a dose of mg/rabbit (farmaki et al., ) . as with psoroptes, more 'natural' treatments are being investigated with good preliminary results from eugenol (pasay et al., ) and eupatorium spp. (nong et al., ) . research complications no specific research complications have been reported. sarcoptes can cause a selflimiting dermatitis in humans. a wide variety of arthropod parasites has been reported in wild rabbits but they are extremely rare in laboratory rabbits. for an extensive listing the reader is referred to other sources (hofing and kraus, ) . etiology historically, the rabbit pinworm was identified as oxyuris ambigua, but is now known as passalurus ambiguus (hofing and kraus, ) . clinical signs even when rabbits have heavy oxyurid burdens, clinical signs are not usually apparent (erikson, ; soulsby, ). one case report described unsatisfactory breeding performance and poor condition in a rabbit colony infested with the parasite. epizootiology p. ambiguus has a direct life cycle. mature pinworms are found in the lumen of the cecum or colon of the rabbit. after ingestion, the eggs hatch in the small intestine, and the larvae molt with maturation in the cecum. the prepatent period is between and days (taffs, ) . transmission occurs easily via ingestion, given that individual rabbits have been found with over adult parasites (hofing and kraus, ) and that embryonated eggs pass out in the feces and are immediately infective (schoeb et al., ; taffs, ) . pathology minimal to no lesions are associated with this pinworm (schoeb et al., ) . diagnosis eggs can be found in feces, cecum, or colon. differential diagnoses this is the only reported pinworm in rabbits and it is not known to cause lesions or disease. treatment, prevention, and control several successful treatment strategies for rabbit oxyuriasis have been reported. piperazine citrate at mg/ ml of drinking water for day was successful in eliminating infestation (hofing and kraus, ) . at and ppm, fenbendazole mixed in the food for days eliminated all immature and adult pinworms (duwell and brech, ) . subcutaneous doses of ivermectin ( . mg/kg) were reported to be ineffective in reducing the burden of passalurus organisms in field populations of snowshoe hares (lepus americanus) (sovell and holmes, ) . due to the direct life cycle, strict husbandry and sanitation practices are required to prevent introduction and spread throughout a rabbit colony (schoeb et al., ) . research complications none have been described. etiology dermatophytosis, also known as ' ringworm' or 'tinea', refers to a skin infection caused by a dermatophyte, a keratinophilic and keratinolytic fungus (chermette et al., ; mendez-tovar, ; robert and pihet, ) . dermatophytes are a group of closely related filamentous fungi that are able to invade the stratum corneum of the epidermis and keratinized tissues including the skin, nail, and hair (kanbe, ) . dermatophytes can infect various animal species, including humans, and the disease is considered contagious and zoonotic (cafarchia et al., b; chermette et al., ; kramer et al., ) . the zoophilic dermatophytes trichophyton mentagrophytes and microsporum canis infect rabbits (cafarchia et al., (cafarchia et al., , a chermette et al., ; kramer et al., ) . clinical signs the general presentation of dermatophytosis in animals is an area of circular alopecia with erythematous margin and thin desquamation (chermette et al., ) . pruritus is generally absent and lesions can be single or multiple (chermette et al., ) . although lesions can be localized in any region, the anterior part of the body and the head seem to be more frequently involved (chermette et al., ) . in rabbits, lesions are often found on the ears and the face (around the eyes and on the nose) and these lesions show scaling and crusting (chermette et al., ; kramer et al., ) . infected rabbits may not exhibit clinical signs and may serve as carriers (balsari et al., ; cafarchia et al., cafarchia et al., , a chermette et al., ; lopez-martinez et al., ) . epizootiology although dermatophytosis is a common cutaneous disease of rabbits and other animals, its incidence is low in well-managed laboratory animal facilities (chermette et al., ; connole et al., ) . contact with infected animals or contaminated environments represent the major risk of infection (chermette et al., ) . young or immunocompromised rabbits are more susceptible (connole et al., ; kramer et al., ) . on rabbit farms, the occurrence of lesions, the age of the rabbits, and farm management practices were identified as the most significant risk factors for the occurrence of dermatophytosis (cafarchia et al., ) . clinically, disease expression varies depending on the host, fungal species, and enzyme production (cafarchia et al., a; vermout et al., ) . the pathogenesis involves contact, adherence, germination, invasion, and penetration (cafarchia et al., a; mendez-tovar, ; vermout et al., ) . these stages can be associated with the secretion of enzymes that degrade the infected tissue components (cafarchia et al., a) . t. mentagrophytes isolates from rabbits with skin lesions showed a significantly higher elastase and gelatinase activity compared laboratory animal medicine to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . furthermore, m. canis isolates from rabbits with skin lesions showed a significantly higher lipase activity compared to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . pathology histopathologic changes consist of mild to severe dermatitis. diagnosis the wood's lamp (ultraviolet light) method and direct examination of hairs and scales are fast and affordable tests (chermette et al., ; robert and pihet, ) . the wood's lamp can be used to screen for infections caused by m. canis (chermette et al., ) . m. canis-infected hairs fluoresce with an apple-green color and can be collected for microscopic examination and culture (chermette et al., ) . the results of the wood's lamp examination should be systematically confirmed by direct examination of hairs and/or fungal culture (chermette et al., ) . deep skin scraping should be performed to obtain hair and scales and confirm the absence of ectoparasites such as mites that can be associated with dermatophytosis (cafarchia et al., ; chermette et al., ) . clearing solutions such as chlorolactophenol or % potassium hydroxide (koh) can then be used to digest keratin prior to microscopic examination (chermette et al., ; robert and pihet, ) . the surface of the hair typically demonstrates clusters or chains of arthroconidia (chermette et al., ) . giemsa-stained skin scrapings allow observation of the arthroconidia along the hair (chermette et al., ) . fungal culture is the 'gold standard' for the diagnosis of dermatophytosis and the only method for the phenotypic identification of dermatophyte species (chermette et al., ) . the fungal culture must be complemented with direct examination of samples for optimal interpretation of the results (chermette et al., ; robert and pihet, ) . samples for fungal culture may include hairs, scales, crusts, skin scrapes, and tissue biopsies (chermette et al., ) . samples that are obtained from the margin of new skin lesions enhance fungal recovery by culture (chermette et al., ) . a brush can also be impressed on the surface of the culture medium after combing the fur and obtaining fungal spores with hair and debris (robert and pihet, ) . two media that can be used for fungal culture include sabouraud dextrose agar (supplemented with cycloheximide and antibiotics) and dermatophyte test media (dtm) (chermette et al., ; robert and pihet, ) . if histological examination is performed, periodic acid schiff (pas), or methylamine silver stain can be used to detect arthroconidia and hyphae (chermette et al., ) . molecular methods to identify dermatophytes have also been described and include pcr-rflp and sequencing of the internal transcribed spacer (its) region (chermette et al., ; kanbe, ; robert and pihet, ) . specific identification of the dermatophyte is essential for a better understanding of the epidemiology and prevention of the disease (chermette et al., ) . differential diagnoses the differential diagnoses can include other dermatoses caused by bacteria or ectoparasites (cafarchia et al., ; chermette et al., ) . treatment, prevention, and control dermatophytosis is considered a self-limiting disease in immunocompetent animals (chermette et al., ) . however, rabbits with dermatophytosis should be culled or separated from a laboratory animal colony due to the contagious and zoonotic nature of the disease (chermette et al., ) . the best method to prevent dermatophyte infection is to prevent contact with infected animals and contaminated environments including fomites (chermette et al., ) . an animal that contacts an infected animal or a contaminated environment can be washed with antifungal shampoo (chermette et al., ) . two vaccines incorporating live attenuated cells of t. mentagrophytes have been used to prevent disease in rabbits and other animals (lund and deboer, ) . the mentavak vaccine is from russia and the trichopelen vaccine (http://www. bioveta.cz/en/veterinary-division/home/) is from the czech republic (lund and deboer, ) . trichopelen is also indicated for treatment of dermatophytosis (lund and deboer, ) . enzootic dermatophytosis may be the result of the high resistance of the arthroconidia in the environment, the number of host species involved, and the close confinement of animals (chermette et al., ) . isolation or culling of infected animals plus environmental and equipment disinfection are required to control this disease (chermette et al., ) . a : dilution of household bleach or a . % enilconazole solution can be used to disinfect the environment (chermette et al., ) . infected animals should be handled with care to avoid zoonotic transmission (chermette et al., ) . if treatment is elected, antifungal treatment shortens the course of the infection and reduces dissemination of arthroconidia to other animals and into the environment (chermette et al., ) . systemic and topical antifungal treatment can be used in combination (chermette et al., ) . systemic drugs include griseofulvin (gold standard) or azole derivatives such as itraconazole (chermette et al., ; vella, ) . it is important to know that these drugs can have side effects and be contraindicated due to their teratogenic potential (chermette et al., ) . topical treatment may include . % enilconazole, a combination of % miconazole and % chlorhexidine, or lime sulfur (chermette et al., ; vella, ) . treatment can be discontinued after two negative fungal culture results (chermette et al., ) . research complications dematophyte lesions could confound histological studies involving the skin (connole et al., ) . etiology pneumocystosis in rabbits is caused by the fungus pneumocystis oryctolagi (dei-cas et al., ) . clinical signs infected rabbits may not develop clinical signs, but immunocompromised hosts can develop severe interstitial pneumonitis (dei-cas et al., ; sheldon, ) . epizootiology corticosteroid treatment can induce disease in infected rabbits; however, spontaneous disease (not associated with drug treatment) can also occur (dei-cas et al., ; sheldon, ; soulez et al., ) . p. oryctolagi is transmitted through the transplacental route (cere et al., a; sanchez et al., ) and through direct contact and aerosolization (cere and polack, ; cere et al., b; hughes, ; wakefield, wakefield, , . spontaneous pneumocystosis can occur at weaning, evolves during - days, and induces lung lesions and blood biochemical profile changes (dei-cas et al., ; soulez et al., ) . the organisms attach specifically to type epithelial alveolar cells and proliferate, filling up pulmonary alveoli cavities leading to respiratory failure (dei-cas et al., ) . changes in surfactant appear to be necessary for pneumocystis proliferation (prevost et al., ) . pneumocystis colonization decreases and becomes very low in -day-old rabbits (dei-cas et al., ) . most rabbits recover from pneumocystosis within - weeks (dei-cas et al., ) . the spontaneous resolution of pneumocystosis in rabbits may be associated with expression of interferon gamma (allaert et al., ) . immunosuppression may be suspected in cases of severe pulmonary disease associated with spontaneous pneumocystosis (sheldon, ) . pathology histologically, cystic forms of the organism can be detected in the lungs using toluidine blue o (tbo), gms, or pas stains (dei-cas et al., ) . interstitial thickening of alveolar septa and increased numbers of type epithelial alveolar cells are characteristic of this infection (creusy et al., ) . diagnosis for diagnosis, samples from nasal cavity wash, or post-mortem, from terminal bronchoalveolar lavage (bal) or lung homogenates can be used for pneumocystis detection by nested pcr (dei-cas et al., ; tamburrini et al., ; wakefield, ) . serological diagnosis can also be performed (tamburrini et al., ) . lung impression smears, lung-homogenate smears, and bal fluid samples can be stained for microscopic detection of pneumocystis (dei-cas et al., ) . useful stains include tbo, gomori-grocott's methenamine silver nitrate (gms), and methanol-giemsa or giemsalike stains (dei-cas et al., ) . other useful detection methods include phase-contrast microscopy and the use of pneumocystis-specific fluorescein-labeled antibodies (dei-cas et al., ) . differential diagnoses p. multocida can induce respiratory disease in rabbits and can be included in the differential diagnoses. treatment, prevention, and control for prevention, new rabbits should be negative for pneumocystis. cotrimoxazole treatment and nested pcr have been used as a screening mechanism to eliminate pneumocystis from colony-maintained rabbits (cere et al., c) . decontamination practices and air filtration were also important for eradication (cere et al., c) . confirmation of a pneumocystis-free status in a rabbit colony was demonstrated by negative pcr results and/ or failure to induce pneumocystosis after experimental corticosteroid challenge (dei-cas et al., ) . research complications research studies may be affected if rabbits of unknown pneumocystis status are experimentally treated with corticosteroids or other immunosuppressant drugs (sheldon, ) . pulmonary lesions may be found in infected rabbits and could potentially confound respiratory research studies (sheldon, ) . etiology unknown. clinical signs trichobezoar is often subclinical. if the trichobezoar causes partial or complete blockage, clinical signs of gastric or intestinal obstruction will result. death can occur due to prolonged anorexia and metabolic imbalances (gillett et al., ). it appears that obstruction of the pylorus, and not the volume of the gastric mass, is the critical factor in determining the clinical progress of the animal (leary et al., ) . epizootiology the condition occurs sporadically in rabbit colonies. pathology the discovery of a hairball in a rabbit is often an incidental finding during necropsy. up to % of rabbits have been found to have gastric trichobezoars during routine necropsy (leary et al., ) . gastric rupture can also result from an obstructive trichobezoar (gillett et al., ) . diagnosis diagnosis is often difficult because the clinical signs are nonspecific and the disease often progresses gradually. some cases involving acute pyloric obstruction result in sudden clinical disease and rapid clinical decline of the animal. manual palpation may indicate the presence of a firm mass in the cranial abdomen. gastric radiographs using contrast media may aid in the diagnosis, but definitive diagnosis is often made during exploratory surgery (gillett et al., ) . differential diagnoses constipation and intestinal foreign body should be considered in the differential list. treatment, prevention, and control treatment of trichobezoar is often unsuccessful. oral administration of mineral oil at ml/day has been reported (suckow and douglas, ) . alternatively, oral administration of - ml of fresh pineapple juice daily has been reported as a possible treatment modality (harkness and wagner, ) . if medical treatment does not resolve the condition, a gastrotomy should be performed. early surgical intervention is important in such cases, as other, subsequent metabolic abnormalities may quickly increase the surgical risk to the rabbit (bergdall and dysko, ) . research complications none have been reported. etiology subluxation or compression fractures of lumbar vertebrae are often secondary to struggling during restraint, particularly when the hindquarters of the rabbit are not supported (bergdall and dysko, ) . the seventh lumbar vertebra (l ) or its caudal articular processes are considered the most frequent sites of fractures, with fracture occurring more commonly than dislocation (flatt et al., ) . clinical signs clinical signs include posterior paresis or paralysis, loss of sensation in the hindlimbs, urinary and/or fecal incontinence, and perineal staining. pathology spinal cord hemorrhage and necrosis can be found. diagnosis diagnosis is based on clinical signs, history of recent restraint, struggling or other trauma, and palpation or radiographic analysis of the vertebral column. differential diagnoses spinal cord trauma. treatment, prevention, and control euthanasia of affected animals is usually warranted. moderate cases (subluxation with spinal edema) may resolve over time. the decision to euthanize should be based on severity of clinical signs. supportive care includes regular expression of the urinary bladder and prevention and treatment of decubital ulcers. corticosteroid and diuretic therapy may be effective for cases of subluxation with spinal edema (bergdall and dysko, ) . research complications loss of valuable research animals is the primary complication. although the condition is often referred to as 'sore hocks,' the correct name is ulcerative pododermatitis. despite the name, the condition rarely affects the hocks, but rather occurs most frequently on the plantar surface of the metatarsal and, to a lesser extent, the metacarpal regions. the condition is believed to be initiated by wire-floor housing, foot stomping, or having thin plantar fur pads. poor sanitation may worsen the condition. solid resting areas on the cage floors are associated with a decreased incidence of ulcerative pododermatitis, whereas a high-energy diet and increased body condition scores are associated with an increased incidence (sanchez et al., ) . the whole genome sequence from a single female rabbit of the partially inbred thorbecke rabbit strain was published in (orycun . ; accession aagw ). the annotated assembly is now available at the national center for biotechnology information (ncbi), the university of california santa cruz (ucsc), and ensembl. the rabbit chosen by the broad institute for sequencing was obtained from covance in . the assembly has . gbp in autosome and x chromosomes and mbp in unplaced scaffolds including mitochondria (gertz et al., ) . the nucleotide sequence of the complete mitochondrial dna (mtdna) molecule of the o. cuniculus has been determined (gissi et al., ) . the compositional differences between the two mtdna strands have also been detailed (gissi et al., ) . the sequencing of the rabbit genome, understanding of rabbit reproduction, and advances in genetic manipulation in the mouse production colonies have led to the ability to produce genetically engineered rabbits. the rabbit offers an alternative model when size or tissue characteristics of a genetically modified mouse are not appropriate. these genetic manipulation techniques were first described by robl (robl and burnside, ) . additional methods have been developed and include pronuclear injection of single cell embryos, injection of genetically modified embryonic stem cells into blastocysts, sperm-mediated gene transfer, and genetically modified somatic cell and nuclear transfer (christensen and peng, ) . commercial companies have been formed to provide genetic modification services with emphasis on production of a unique protein in the milk of rabbits. this section will outline spontaneous hereditary conditions of the rabbit that have been well characterized. some conditions represent conditions that have been identified in humans and other conditions offer insight into the mechanism(s) of particular organ or immune function. hydrocephalus refers to dilatation of the cerebral ventricles and is usually accompanied by accumulation of cerebrospinal fluid within the dilated spaces. some cases of hydrocephalus in rabbits have been presumed to be related to a single autosomal recessive gene (hy/hy); however, occurrence with other abnormalities suggests that inheritance may be more complicated (lindsey and fox, ) . in some cases, the condition appears to be inherited along with various ocular anomalies as an autosomal gene with incomplete dominance. hydrocephalus may also occur in rabbits as a congenital condition related to hypovitaminosis a in pregnant does (lindsey and fox, ) . etiology buphthalmia is inherited as an autosomal recessive trait, although penetrance is presumably incomplete since severity and the age of onset vary greatly and some bu/bu individuals do not develop buphthalmia (hanna et al., ) . clinical signs rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. newborn bu/bu rabbits initially have normal intraocular pressure (iop; - mmhg) but increased pressures of - mmhg may develop after - months of age (burrows et al., ; knepper et al., ) . the eyes become progressively buphthalmic (either uni-or bilaterally) but the iop can return to normal or to sub-normal levels after - months. typical clinical changes include increased corneal diameter as the globe enlarges because the sclera is still immature. the cornea may develop a cloudy or bluish tint, corneal edema, increased corneal vascularity, and flattening of the cornea. structural changes may include widening of the angle, thickening of descmet's membrane, atrophy of the ciliary process, and excavation of the optic disk. impaired aqueous outflow may be due to incomplete cleavage of the drainage angle with abnormal insertion of uveal tissue into the cornea (tesluk et al., ) . in some cases, the cornea ulcerates and ruptures. there is also a marked reduction in semen concentration in buphthalmics, with a decrease in libido and decreased spermatogenesis in affected males (fox et al., ) . epizootiology the condition is common in new zealand white rabbits. pathology by weeks of age, the morphology of the congenital glaucoma trabecular network becomes abnormal with a smaller entrance to the trabecular network at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and by weeks, iris pillars with extensive lateral extensions in the angle recess can be observed. most intertrabecular spaces remain open; however, the inner intertrabecular spaces adjacent to the aqueous plexus become compressed. diagnosis diagnosis is based on clinical signs and measurement of intraocular pressure. treatment, prevention, and control specific treatment of buphthalmia has not been described for rabbits; however, affected individuals should not be used for breeding purposes. research complications loss of valuable research animals is the primary complication. etiology mandibular prognathism is the most common inherited disease of domestic rabbits. the condition is inherited as an autosomal recessive trait (mp/mp) with incomplete penetrance (fox and crary, ; huang et al., ; lindsey and fox, ) . clinical signs malocclusion related to mandibular prognathia may be clinically apparent as early as - weeks of age, but is more typically seen in older rabbits post weaning. clinical signs may include anorexia and weight loss. if severe enough and left untreated, affected animals will starve since they cannot properly prehend and masticate food. epizootiology normally, the lower incisors occlude with the large upper incisors, as well as with a pair of small secondary incisors that are immediately caudal to the primary maxillary incisors. the lower set of incisors typically wear against the upper set during normal biting activity, along an arc formed by biting movements of the lower incisors, whereas the maxillary secondary incisors wear at right angles to the mandibular incisors. the incisors wear more quickly at the posterior aspect in rabbits, partly because the enamel layer is thinner on that side. affected rabbits have a normal dental formula. the specific abnormality associated with mandibular prognathism is that the maxilla is short relative to a mandible of normal length. thus, although the mandible appears abnormally long, the primary defect involves the maxilla. in rabbits, the teeth (including the molars and premolars) grow continuously throughout life. the incisors, for example, grow at the rate of . - . mm/ week. when occlusion is normal, the teeth wear against one another and in this way remain a normal length. however, when occlusion is abnormal because of conditions that include mandibular prognathia, the teeth may become greatly elongated because typical attrition of the incisors does not occur. in affected animals the lower incisors often extend anterior to the upper incisors and protrude from the mouth, whereas the upper primary incisors grow past the lower incisors and curl within the mouth. in some instances, the upper incisors curl around dorsally and lacerate the mucosa of the hard palate. secondary infection and abscessation may occur in such cases. diagnosis diagnosis is based on clinical signs. differential diagnoses malocclusion secondary to mandibular or maxillary fracture should be considered. treatment, prevention, and control overgrown teeth should be trimmed every - weeks or more frequently if needed. trimming is preferably performed with a dental bur to avoid cracking the tooth, which laboratory animal medicine may happen more frequently if a bone or wire cutter is used. care should be taken to avoid exposing the pulp cavity as the result of excessive trimming. because the condition is hereditary, use of affected animals as breeding stock should be avoided. research complications no specific research complications have been reported. a number of disorders characterized clinically by complete abduction of one or more legs and the inability to assume a normal standing position are described by the term 'splay leg'. young kits of - weeks of age are most commonly affected. affected rabbits cannot adduct limbs and have difficulty in making normal locomotory movements. most commonly, animals are affected in the right rear limb, although the condition may be uni-or bilateral and may affect the anterior, posterior, or all four limbs. rabbits with splay leg may have difficulty in accessing food and water; thus, attention to adequate nutrition is required as part of proper clinical care. the clinical signs of splay leg may be due to an overall imbalance of development of the neural, muscular, and skeletal systems. possibly, some animals compensate with torsion and exorotation of the limb at the hip, whereas rabbits that are unable to compensate are clinically affected. although the precise pathogenesis of splay leg is not entirely understood, at least some cases are ascribed to inherited disorders. typical clinical signs are secondary to femoral endotorsion, with a shallow acetabulum but without luxation of the femur at the hip. the semitendinosus muscle of affected animals is abnormal, with smaller fibers and abnormal mitochondria. some reports suggest that the condition is associated with inherited achondroplasia of the hip and shoulder, whereas others indicate that a recessively inherited anteversion of the femoral head can be involved. self-mutilating behavior in a checkered cross (cross between english spot, german checkered giant, and checkered of rhineland rabbits) was reported to occur as an inherited trait (iglauer et al., ) . autotraumatization of the feet and pads was observed. the abnormal behavior could be interrupted by administration of haloperidol. although not manifested as a disease, the presence of serum atropine esterase allows rabbits to inactivate atropine when administered for therapeutic purposes (liebenberg and linn, ; stormont and suzuki, ) . the enzyme also permits rabbits to consume diets containing belladonna compounds. the enzyme is produced by a semidominant gene est- f. three phenotypes are recognized depending on the number of genes expressed. the enzyme first appears in the serum at month of age, and enzyme levels are greater in females than in males (lindsey and fox, ) . the est- f gene is linked to genes controlling the black pigment in the coat (forster and hannafin, ; fox and van zutphen, ; sawin and crary, ) . hereditary deficiency of the third component of complement (c ) was found in a strain of rabbits. this same strain also exhibited a hereditary c alpha-gamma deficiency. the serum c concentration, hemolytic c activity, and total complement hemolytic activity of these animals were significantly reduced. the low level of serum c in these rabbits was not due to c conversion, partial c antigenicity, and presence of a c inhibitor or hypercatabolism of normal c . the c deficiency was transmitted as a simple autosomal co-dominant trait. rabbits with this trait have a lower survival at months than normal rabbits (komatsu et al., ) . this complement deficiency syndrome in the rabbit has been well characterized. this syndrome was initially reported in in a strain of rabbits that lacked the sixth component of the hemolytic complement system (rother et al., ) . whole blood clotting time in glass or plastic was prolonged and prothrombin consumption was decreased in blood from the deficient animals. other parameters of blood coagulation were normal, including prothrombin time, partial thromboplastin time, specific clotting factor activities, platelet factor iii function, platelet count, and bleeding time (zimmerman et al., ) . abnormal platelet response is also characteristic of this syndrome in the rabbit (lee et al., ) . complement c -deficient rabbits are protected against diet-induced atherosclerosis despite having similar profiles in cholesterol levels and plasma lipoprotein. when compared to normal rabbits, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all normal rabbits while absent in all c -deficient animals (schmiedt et al., ) . the inheritance pattern for this defect is autosomal recessive (abe et al., ) . a progressive neurological syndrome has also been observed in the c -deficient rabbits. this syndrome is clinically characterized by subacute motor neuropathy. pathological studies of affected animals revealed ( ) severe axonal degeneration in the sciatic nerve involving mainly motor fibers; ( ) occasional peripheral axonal enlargement closely associated with axonal laboratory animal medicine degeneration; ( ) presence of structured abnormal material in normal-size myelinated fibers of the central and peripheral nervous systems; and ( ) widespread occurrence of dystrophic axons and axonal spheroids in the gray matter of the central nervous system. by ultrastructural examination, dystrophic axons were filled with tubulovesicular material, appearing as stalks of parallel membranes and dense bodies similar to what is described in human neuroaxonal dystrophies (nad). the disease manifested by c -deficient rabbits may represent an animal model of primary human nad (giannini et al., ) . genetic deficiency of the alpha-gamma-subunit of the eighth complement component (c alpha-gamma) was found in a substrain of the new zealand white rabbits. the serum of this deficient rabbit lacked the immunochemical and functional alpha-gamma-subunit of c (c d). this syndrome is transmitted as a simple autosomal recessive trait. the syndrome is characterized by smaller body weight compared to those of heterozygous and normal rabbits. in addition, survival rates for the first months of life of the deficient animals tended to be lower than those of heterozygous and normal littermates (komatsu et al., ) . all c d rabbits (more than animals obtained thus far) were consistently smaller than normal littermates from birth to adulthood, i.e., % of normal size at birth, % of normal size at days of age, and % of normal size at adulthood. the c α-γ deficiency in rabbits is always associated with dwarfism. furthermore, there appears to be a discrete recessive dwarf gene (dw- ), whose locus is not linked to c d. rabbits double-homozygous for c d and dw- (severe dwarf) were smaller than the c d or dwarf rabbits and almost all of the severe dwarf rabbits died within days after birth. the actual and relative weights of the thymus in the c d rabbits were consistently lower than those of normal rabbits, but histological examination of the c d thymus did not reveal any abnormalities. the c d and dwarf rabbits were fertile; however, crosses of c d females with c d or dwarf males led to a reduced delivery rate and small litter size. the c d locus is loosely linked to the c hypocomplementemic locus (c -hypo) (map distance cm) but not to the hemoglobin blood group locus (komatsu et al., ) . the inherited characteristics of the kurosawa and kusanagi hypercholesterolemic (khc) rabbit include persistent hypercholesterolemia. this strain of rabbits was produced by inbreeding mutants discovered in . these khc rabbits had serum cholesterol, triglyceride, and phospholipid levels - times greater than clinically normal o. cuniculus. the khc rabbits also had decreased serum high-density lipoprotein cholesterol concentration, about one-third the value in clinically normal rabbits. in addition, the serum lipoprotein electrophoretic patterns were characterized by a strong, broad beta-lipoprotein band and a diminished alphalipoprotein band. fractionation of lipoprotein lipids revealed increased cholesterol, phospholipid, and triglyceride in the ldl fraction; increased cholesterol and phospholipid in the very ldl fraction; and decreased cholesterol and triglyceride in the high-density lipoprotein fraction. the inheritance is thought to be a single autosomal recessive gene mutation, and analysis of the ldl receptor indicated that the khc rabbit has a -base pair deletion in the ldl receptor mrna. macroscopic analysis of the aorta revealed the atheromatous lesions at months of age, drastically increased lesional areas in the total aortic surface at months of age, and a high incidence of coronary atheromas and xanthomas (kurosawa et al., ) . a spontaneous phenotype in a rabbit was discovered with an elevation of serous lipid ingredients including cholesterol and beta-lipoprotein (beta-lp). atherosclerotic lesions were evident in the aorta and renal arteries. nodular xanthomas were also present on the front and rear feet. the hlr strain was inbred to accentuate these characteristics (watanabe et al., ) . the strain was eventually designated the watanabe-heritable hyperlipidemic rabbit (whhl-rabbit). an additional report of this strain of rabbits indicated that the whhl-rabbits spontaneous developed aortic atherosclerosis by months of age and xanthoma of digital joints in % of the rabbits aged to months (watanabe, ) . historically, spontaneous neoplasia in the laboratory rabbit has not been widely reported because neoplasia in the rabbit is very uncommon before years of age and many laboratory rabbits are not maintained beyond this age (weisbroth, ) . endometrial adenocarcinoma is the most common tumor in aged female rabbits, with an incidence of % reported in a colony of -year-old rabbits (baba and von haam, ) . tinkey et al. complied an extensive review of the literature dealing with spontaneous neoplasia in the domestic rabbit. this review contained data on case reports, descriptions of biologic aspects of naturally occurring tumors and reports of experimentally induced tumor models. neoplasia in sylvilagus and lepus were also discussed (tinkey et al., ) . uterine adenocarcinoma is by far the most common tumor in rabbits. typically, the disease is present as multiple tumors and is malignant, often metastasizing to the liver, lungs, and other organs. there is evidence that inheritance plays a role in susceptibility, but parity does not. uterine leiomyomas and leiomyosarcomas are much less common (weisbroth, ) . there are a few reports of vaginal squamous cell carcinomas (weisbroth, ) and an ovarian hemangioma has been described (greene and strauss, ) . mammary adenocarcinomas are fairly common in older female rabbits and may occur in animals with uterine adenocarcinoma (weisbroth, ) . papillomas have been described, but mammary adenocarcinomas are much more important. these malignant tumors may metastasize, but the cause of death in affected rabbits is often due to uterine adenocarcinoma. serial biopsy studies indicate that these tumors are preceded by cystic mastopathy as well as changes in the adrenal and pituitary glands (greene, ) . there may also be small prolactin-secreting pituitary adenomas in rabbits with mammary dysplasia (lipman et al., ) . testicular tumors in the rabbit appear to be relatively uncommon. interstitial tumors are the most common testicular tumor in the rabbit. seminomas and teratomas have also been reported (weisbroth, ) . embryonal nephromas are one of the most common tumors in laboratory rabbits. these tumors are often found incidentally, occur in younger animals, and seldom cause clinical signs (weisbroth, ) . there has been one report of a renal carcinoma in the rabbit (kaufman and quist, ) and one report of a leiomyoma arising in the urinary bladder (weisbroth, ) . malignant lymphomas (lymphosarcomas) are relatively common in rabbits. they may occur in rabbits that are less than years of age (weisbroth, ) , but older rabbits may also be affected. according to (weisbroth, ) , a tetrad of lesions is often seen. these lesions include enlarged kidneys, splenomegaly, hepatomegaly, and lymphadenopathy. older rabbits have presented with skin nodules and eye lesions; however, malignant lymphomas in the rabbit are seldom leukemic. most cases of malignant lymphoma appear to resemble the lymphoblastic subtype as seen in humans and mice. malignant lymphoma is more prevalent in some strains of rabbits than in others, and there is some evidence for a retroviral cause of lymphomas in rabbits (weisbroth, ) . true thymomas (containing both lymphoid and epithelial components) (vernau et al., ) and plasma cell myelomas (pascal, ) are rare in rabbits. one case of myeloid leukemia has been reported (meier et al., ) . basal cell tumors are reported to be rare (weisbroth, ) , but they may be underreported (li and schlafer, ) . squamous cell carcinomas are also uncommon, and there is no apparent predilection for any particular area of the body (weisbroth, ) . other cited skin-associated tumors include a trichoepithelioma (altman et al., ) , a sebaceous gland carcinoma (port and sidor, ) , and two malignant melanomas (hotchkiss et al., ) . osteosarcomas are extremely rare in rabbits, and most have arisen in the mandible or maxilla, with only one found in a long bone (weisbroth, ) . no primary tumors arising in cartilage have been described, although some of the reported osteosarcomas have had cartilaginous elements. one tumor of skeletal muscle, a rhabdomyosarcoma, has been reported. a few fibrosarcomas and one fibrosarcoma involving the foot have been reported (weisbroth, ) . a number of case reports of single tumors are found in the literature. these include a peritoneal mesothelioma (lichtensteiger and leathers, ) , an intracranial teratoma (bishop, ) , an ependymoma (kinkier and jepsen, ) , a neurofibrosarcoma, two hemangiosarcomas (pletcher and murphy, ) , and a malignant fibrous histiocytoma (yamamoto and fujishiro, ). there are a few very old reports of lung tumors dating to the first part of the th century (weisbroth, ) . there are several tumor models in which the cells used for inoculation were originally derived from rabbit tumors. these include the vx- carcinoma (kidd and rous, ) , the brown pearce carcinoma (brown and pearce, ) , and the greene melanoma (greene, ) . the vx- carcinoma originated from a squamous cell carcinoma in a rabbit carrying a shope papilloma. the most common modern use of this transplantable tumor is as a model for the study of various cancer treatment modalities for metastatic tumors (stetson et al., ) . the brown pearce carcinoma arose from a tumor in a rabbit testis, but the exact tissue of origin of the tumor was never determined. the tumor was readily transplantable and caused stable metastases. because some tumors regress, even after widespread metastases, this tumor has been used as a model for the study of tumor immunology (weisbroth, ) . the brown pearce laboratory animal medicine carcinoma, although extensively characterized and historically used, has been reported in the literature only five times from to (tinkey et al., ) . hydrometra has been described as a clinical condition of rabbits. all cases were in unmated rabbits that were used experimentally for the production of serum antibodies (bray et al., ; hobbs and parker, ; morrell, ) . clinical signs included abdominal distension and tachypnea. cases were characterized by distension of the uterine horns with a transudative fluid. one case was associated with uterine torsion (hobbs and parker, ) . one case had resolved with diuretic therapy, only to return later (bray et al., ) . most cases of liver lobe torsion in rabbits involve the caudate lobe (bergdall and dysko, ) , although one case report described torsion of the left hepatic lobe (wilson et al., ) . most reported cases have been incidental findings at necropsy. incidental hepatic lobe torsions have also been identified in three adult new zealand white rabbits that died from pasteurellosis (weisbroth, ) . three cases of hepatic torsion in pet rabbits were reported by wenger in (wenger et al., ) . all rabbits presented with an acute onset of lethargy, anorexia, abdominal pain, pale mucous membranes, and jaundice. one rabbit also had hematuria. another report of caudate liver lobe torsion also described a rabbit that was jaundiced, anemic, and anorexic, with elevated alanine aminotransferase. torsion of the caudate liver lobe was seen at necropsy (fitzgerald and fitzgerald, ) . in all reported clinical cases, rabbits were euthanized, or died during postoperative recovery. calcium carbonate and triple phosphate crystals are present in the urine of normal rabbits. these crystals contribute to the cloudy consistency of the urine (williams, ) . a -year retrospective study of hematuria in new zealand white rabbits was conducted by garibaldi (garibaldi et al., ) . physical examination, laboratory tests, radiography, and postmortem examination were utilized in most cases to verify the presence of hematuria and to determine its etiology. uterine adenocarcinoma was diagnosed in two rabbits. three rabbits had uterine polyps with hemorrhage. renal infarction with hemorrhage was diagnosed in three rabbits. urolithiasis with secondary urethral obstruction and hemorrhagic cystitis was identified as the cause of hematuria in four rabbits. other causes of hematuria included chronic cystitis, disseminated intravascular coagulation, bladder polyps and pyelonephritis. hematuria of undetermined origin was observed in one rabbit which emphasizes that hyperpigmented urine should be a rule out in all cases of suspected hematuria in rabbits (garibaldi et al., ) . one case of urolithiasis with hydronephrosis in a new zealand white rabbit was also reported (labranche and renegar, ) . this condition must be distinguished from hematuria caused by endometrial venous aneurysm in female rabbits (bray et al., ) . herniation of the kidney along with perinephric fat has been reported (suckow and grigdesby, ) . the affected rabbit was clinically normal except for a subcutaneous mass that had passed through the body wall. the precise etiology is not known, although it was speculated that herniation might have occurred as the result of unreported trauma. occlusion of the nasolacrimal duct, presumably due to accumulation of fat droplets, has been described as a putative cause of epiphora in some rabbits (marini et al., ) . although the obstruction occurred at the dorsal flexure, it is not clear if this was due to congenital rather than acquired stenosis. in a retrospective study of rabbits it was determined that the mean age of the rabbits presenting with ocular discharge from the nasolacrimal duct was . years. in rabbits ( %), dacryocystitis was a unilateral finding. no underlying cause could be determined in animals ( %). dental malocclusion was observed in rabbits ( %) and rhinitis in two animals ( %), with one animal showing both signs ( %). one rabbit ( %) presented with panophthalmitis. most animals ( %) received topical antibiotic treatment. regarding the clinical outcome, animals ( %) showed complete recovery, eight rabbits ( %) were euthanized, three ( %) died due to unrelated causes, and three ( %) were lost to follow-up. two rabbits ( %) continued to display signs of dacryocystitis (florin et al., ) . development and genetic differences of complement activity in rabbits influence of pasteurella multocida infection on 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rabbit: a new model for monitoring the pluripotency of rabbit stem cells flow-volume curves of excised right and left rabbit lungs biochemical parameters of normal rabbit serum a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia a blood coagulation abnormality in rabbits deficient in the sixth component of complement (c ) and its correction by purified c origin of complex behaviour of spatially discordant alternans in a transgenic rabbit model of type long qt syndrome key: cord- -eu gn bl authors: stojanov, ana; bering, jesse m.; halberstadt, jamin title: does perceived lack of control lead to conspiracy theory beliefs? findings from an online mturk sample date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: eu gn bl it is widely believed that conspiracy theory beliefs are the product of perceived lack of control. however, to date there is mixed evidence, at best, to support this claim. we consider the reasons why conspiracy theory beliefs do not appear to be based in any straightforward way on control beliefs, interrogating existing findings and presenting new data that call the relationship into question. across six studies conducted online using mturk samples, we observed no effect of control manipulations on conspiracy theory beliefs, while replicating previously reported correlational evidence of their association. the results suggest that conspiracy beliefs are not suitable for compensating for threats to control. we discuss possible reasons for the discrepancy between experimental and correlational effects and examine the limitations of the studies. the world is ruled by a secret cabal. the us government was involved in the / attacks. hiv is a man-made virus. such statements, which are promoted on the internet and elsewhere, are examples of conspiracy theories: implausible, unwarranted claims that important social events are caused by malevolent clandestine groups, that usually run in contradiction to the explanations offered by the relevant epistemic authorities, and that are embedded in a more general worldview [ ] . although researchers have tried to understand conspiracy theory beliefs-which to nonbelievers appear unwarranted by evidence, if not entirely irrational-from many perspectives, including personality theory [ , ] , intergroup identity [ ] [ ] [ ] , and cognitive bias [ ] [ ] [ ] , arguably the most frequent accounts rely on some notion of "control." the claim that perceived lack of control is the natural breeding ground for conspiracy theory beliefs is a recurring theme in popular science (e.g. [ ] [ ] [ ] [ ] [ ] ), and many academics agree (e.g., [ ] [ ] [ ] [ ] ). however, on closer inspection, neither the theoretical role of control in conspiratorial thinking, nor the empirical relationship between the two constructs, is entirely convincing. although specific accounts differ in their emphasis, control accounts of conspiracy theories generally assume that the perceived loss of personal control is threatening to one's need for order (e.g., [ ] ) or one's schemas about the world (e.g., [ ] ). conspiracy beliefs help compensate for these threats by ascribing control to external entities or organizations (e.g. [ ] [ ] [ ] ), or by creating new meaning frameworks via novel connections among unrelated events. for example, perceiving a connection between a tornado and the unusually long contrails observed the previous day, provides a basis for believing that the government (or some other entity) has deliberately sprayed chemicals in the sky to cause the tornado. however, it seems equally plausible that conspiracy theories would have the opposite effect on the perception of control and order. a common theme among conspiracy theories is that reality is not what it seems; they are counter-normative narratives that suggest institutions and explanations that we take for granted are illusions-a revelation that, if taken seriously, seems bound to challenge the larger meaning-making systems individuals have come to rely on. furthermore, although people desire order, they seek to optimize, not to maximize it, and it could be argued that conspiracy theories provide "too much" order, structure, and meaning by accounting for errant data, connecting seemingly unconnected events and leaving nothing unexplained [ , ] . finally, even if conspiracy theories represent an effective means of restoring order and structure, they are not the only means [ , [ ] [ ] [ ] , and, it would seem, a means of last resort. while control accounts do not require that sources of control or meaning be benevolent, people theoretically favour control systems that are both "culturally accessible" and "socially acceptable" [ , ] . in most-if not all-conspiracy theories, however, the alleged conspirators are malevolent [ ] , thus making them poor candidates for compensatory processes when numerous effective and socially sanctioned alternatives exist. the experimental evidence for conspiracies as compensatory sources of personal control is as ambivalent as its theoretical rationale. in a typical experiment, researchers ask participants to write about a time they did not have control, and to compare these participants' conspiracy beliefs with a control group that is asked to describe an event when they felt in complete control (or about a neutral event). some studies report stronger conspiracy beliefs in the low/neutral control group compared to the high control group (e.g. [ ] [ ] [ ] ), but others report weaker ones (e.g. [ ] ), and yet others (e.g. [ , ] ) report no effects of control on conspiracy beliefs at all. one factor in the variability of results is the corresponding variability-and sometimes dubious validity-of how belief in conspiracy theories is operationalized and measured. for example, whitson and galinsky [ , study ] found that participants recalling uncontrollable situations were more likely to perceive patterns in ambiguous stimuli (e.g., images in visual noise) and links between events (e.g., an increase in e-mail correspondence and a lack of a promotion). however, while conspiracy theories are related to illusory pattern perception [ ] , they are not reducible to it. and while novel connections between seemingly unrelated events can, of course, be the building blocks of conspiracy theories, such theories tell a more complex story that taps into a broader worldview. for example, seeing a connection between the "umbrella man" (a man who opened and lifted an umbrella when kennedy's limousine approached) and the assassination of kennedy is a starting point for building a conspiracy theory, but it is not a conspiracy theory per se (e.g., that kennedy's assignation was an organized soviet-backed plot carried out for political purposes). in another operationalization of conspiracy belief, van prooijen and acker [ ] asked participants about the construction of the north-south metro line in amsterdam, by posing situations that were technically conspiracies, but also plausible abuses of power warranting legitimate skepticism. accusations such as, "the city council transferred parts of the budget to the bank accounts of others", and "members of the city council received money from construction companies to set this plan in motion," describe political corruption, which differ from "conspiracy theories" in scope, plausibility, official endorsement, and psychological interest. indeed, recent evidence indicates that belief in conspiracy theories and belief in corruption are two distinct psychological constructs, predicted by different aspects of a larger construct ("conspiracy mentality"; [ ] ). even studies that explicitly ask about beliefs in specific conspiracy theories may not be well suited for assessing the construct of conspiracy theory belief. for one, participants can easily recognize such items as "conspiracy theories," whose negative connotations are likely to produce socially desirable responses [ ] . moreover, using specific conspiracy beliefs limits the generalizability of the findings to very particular and culturally specific claims, and may say little about a person's general tendency to employ conspiratorial logic. more relevant, we argue, is an individual's tendency to engage in conspiracy thinking, rather than their belief in any particular claim. given the significance and potential consequences of widespread conspiracy beliefs [ , ] , and the plausible but largely unsubstantiated role of control in their appeal, we here report three studies to test the effects of lack of control on conspiracy theory beliefs using a standard priming paradigm and a validated measure of conspiracy ideation, which reflects the belief that a powerful entity lies behind significant social or political events and that the conventional (official) truth is not the "real" truth. a fourth study confirms the results with a measure of specific claims, and two final studies examine two of the most likely moderators of control effects: the degree of control that a particular conspiracy claim affords; and the effectiveness of alternative means of restoring control. although we conceptually replicate previous correlational work-lower control beliefs were associated with stronger conspiracy ideation-we find no evidence for a causal effect. this and subsequent studies were approved by the university of otago human ethics committee. before the beginning of the study participants read an information page and gave written consent for participation; after the study they were debriefed online. participants. in this and subsequent studies, sample size was determined a priori. detecting an effect of f = . , the average effect size reported in psychology research [ ] with alpha = . and power of %, required participants. expecting some attrition, we recruited mechanical turk (mturk) workers ( male, female, other), who completed an online questionnaire designed and presented in the qualtrics survey environment. the mean age of the sample was m = . (range - years, sd = . ). the majority had bachelor's degree ( . %), while . % had a high school degree, . % master's degree, % doctoral degree, . % no degree; . % reported their education level as "other". materials and procedure. participants first read a general information sheet, which explained the tasks that they would be completing, but did not reveal the hypotheses being tested; the passive deception was revealed and explained in post-experimental debriefing. the experiment was described as a study of memory, in which, participants would be asked to recall and describe an event twice, once at the beginning of the study and once "after a break interval," during which they completed the key dependent variables. following whitson and galinsky [ ] , we used the to-be-recalled event as our manipulation of control. the low control group described an event in which they "did not have any control over a situation," while the high control group described an event in which they "were in complete control of the situation." both groups were instructed to include "what happened, how you felt, etc.," and to write at least six lines of text. a neutral group was asked to recall and write about their dinner the previous night. the wording of the low and high control manipulation was taken verbatim from whitson and galinsky [ ] with the exception of the instruction for the length of the text. to assess the effectiveness of the manipulation we measured participants' feelings of control on pearlin and schooler's [ ] mastery scale. it consists of seven items (e.g., i have little control over the things that happen to me) that capture the extent to which people see themselves as being in control over their life [ ] , and which has been used in numerous studies as a measure of perceived control [ , ] . the scale was anchored at (strongly disagree) and (strongly agree). five items were reverse scored, such that higher scores indicate higher levels of control. in the current study cronbach's alpha was . . conspiracy beliefs were measured on the conspiracy theory ideation subscale of stojanov and halberstadt's [ ] conspiracy mentality scale. the subscale consists of seven items capturing the abstract premises of conspiratorial thinking, such as the belief in a powerful entity that controls significant social or political events. (a second dimension of the scale, measuring rational scepticism, is not relevant to the current study.) the subscale has good psychometric characteristics (in the current sample cronbach's alpha was . ) and convergent, divergent and predictive validity, and its construct validity has been confirmed in united states, new zealand and north macedonia populations [ ] . participants rated their agreement with each statement on a -to- scale anchored at "strongly disagree" and "strongly agree." each subscale was presented in a separate block, always in the same order (with the dimension of interest, conspiracy theory ideation, presented first). an attention check item ("to make sure you read attentively please select strongly agree"), was included in the second block. after completing the dependent measure, participants were asked, in line with the cover story, to describe their event a second time (memory was not analyzed). finally, they were asked to speculate about the study's hypotheses, to answer basic demographic questions, and to assess the quality of their data ("did you honestly answer the question in this survey? you'll be paid regardless of how you answer"), before being debriefed. we report all measures, manipulations, and exclusions in this and subsequent studies. twenty-nine participants were excluded from the analysis: sixteen because they failed the attention check question (low control = , neutral = , high control = ); two because they assessed their data as unreliable (high control = ) and (low control = , high control = ) because they expressed suspicion about the research aims (in studies - , two research assistants coded whether the participants were naïve to the research aims. the inter-rater reliability ranged from . to . , and disagreements were settled by the first author) leaving participants in total (including such participants does not change the pattern of results in any of the studies reported in this paper). a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = . . the effectiveness of the control manipulation was tested with a one-way anova on mastery scores, which revealed a significant effect of experimental condition f ( , ) = . , p < . , f = . . post hoc bonferroni comparisons indicated that the low control group (m = . , sd = . , n = ) differed from both the neutral (m = . , sd = . , p < . , n = ) and the high control group (m = . , sd = . , p = . , n = ), but the high control group did not differ from the neutral group (p = . ). this is in line with other unsuccessful attempts to induce higher feelings of control compared to baseline levels [ , ] . contrary to the primary hypothesis, an anova on conspiracy ideation indicated that the low control group (m = . , sd = . ) had descriptively lower conspiracy beliefs than both the neutral (m = . , sd = . ) and high control groups (m = . , sd = . ), although the effect was not significant, f ( , ) = . , p = . , f = . . excluding the high control group (in which the manipulation was ineffective), revealed a marginally significant difference t ( ) = - . , p = . , d = . ; the low and high groups did not differ, t ( ) = - . , p = . , d = . . finally, we calculated the pearson's correlation coefficient between conspiracy beliefs and the mastery score, finding a significant negative relationship, r = - . , p < . . in sum, despite using a standard and empirically successful manipulation of personal control [ , , ] , we did not find that those whose control was threatened expressed greater conspiracy ideation; if anything, the tendency was for the opposite to be true. however, weaker feelings of control (independent of experimental condition) predicted stronger conspiracy ideation, as in previous research [ , , ] . in study , we attempted to replicate the results of study with a conceptually similar but arguably stronger manipulation of control. participants. three hundred mturk participants ( male, female) completed a questionnaire in the online survey platform qualtrics. the mean age of the sample was . years (sd = . , age range - years). the majority had an undergraduate degree ( %), a third ( . %) had a high school degree, . % had a graduate degree, . % other and . % no degree. none of the participants had taken part in study . procedure and materials. after providing informed consent, participants were randomly assigned to one of three groups. those in the high control group were informed that we were interested in the types of situations that people experience as controllable (or in the low control group, as uncontrollable). for additional clarity, the instructions defined the construct of control, noting that "a person is 'in control' of something to the extent that they are able to direct or influence it if they want to." participants were asked to take a moment to think about some of the situations in their life where they have control (or no control), and to briefly describe ten such situations in a separate box. after providing ten examples they were administered the mastery scale as a manipulation check (see study ). the neutral group proceeded immediately to this scale without engaging in any recall activity. as in study , after completing the dependent measures, participants were invited to speculate about the experimental hypothesis before providing demographics and assessing their data quality. twenty-nine participants were excluded from analysis: nine (low control = , neutral = , high control = ) because they failed the attention check question, one (low control) because they self-assessed their data as unreliable and (low control = , high control = , neutral = ) because they were not naïve to the research aims. a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = . . next, we checked the effectiveness of the manipulation. a one-way anova revealed a marginally significant effect, f ( , ) = . , p = . , f = . . ( , ) = . , p = . , f = . . as in study , there was significant negative correlation between the mastery and conspiracy beliefs scores, r = - . , p < . . in sum, despite successfully (albeit weakly) manipulating control and using a conceptually strong measure of conspiracy thinking, neither of two well-powered studies detected any causal evidence for the compensatory hypothesis, though both revealed a correlational relationship. it is worth considering, however, whether the manipulation check itself might be contributing to the null findings, by providing an opportunity to restore control, and thereby eschewing participants' need for further efforts in the form of conspiracy belief change. hauser, ellsworth and gonzalez [ ] argue that manipulation checks as simple as asking participants about their feelings of control may serves the purpose of restoring their control. we note that, if this were true, it supports our contention that there are far easier ways to restore control than to endorse extreme, counter-normative belief systems. nevertheless, we considered this possibility in study . if the presence of the self-report measure of control was responsible for the null effect of the control manipulation on conspiracy ideation, then removing the manipulation check should produce the hypothesized relationship. participants. two hundred and two mturk workers ( males, females) participated. half ( . %) had undergraduate degree, a third ( . %) high school degree, an eighth ( . %) had a graduate degree, while . % selected "other" as their highest level of education, and % reported they did not have a degree. the mean age of the sample was . years (sd = . , range - years). no participants had taken part in previous studies reported in this paper. procedure and materials. the procedure was identical to study , except that the manipulation check was omitted from the procedure, and that control was only manipulated downward. seven participants were excluded from the analysis: one (neutral group) because they self-assessed their data as unreliable, four (neutral = , low control = ) because they failed the attention check question and two (both low control group) because they were not naïve to the research aims. a sensitivity analysis, indicated that the study is able to detect a minimum effect size of f = . . the low control group scored numerically higher on the conspiracy theory ideation (m = . , sd = . , n = ) than the neutral group (m = . , sd = . , n = ), but the difference was not significant, t ( ) = . , p = . , f = . . thus, it does not appear that the inclusion of the manipulation check interfered with participants' hypothesized motivation to restore control via conspiratorial thinking. studies - , using a novel (but, we argue, more theoretically appropriate) operationalization of conspiracy beliefs, revealed no empirical support for the control hypothesis. to explore whether similar results would be obtained with a more typical operationalization of conspiracy beliefs (i.e. as specific claims), and therefore exclude the possibility that the null effects are unique to the conspiracy ideation scale, we replicated the studies again, this time using the beliefs in conspiracy theories inventory [ ] . participants. two hundred mturk workers ( male, female, other) participated. a third had an undergraduate degree ( . %), a third a high school degree ( . %), while . % had a graduate degree, . % did not have a degree, and % had an "other" degree. the mean age of the sample was . years (sd = . , range - years). no participant had taken part in the previous studies reported here. procedure and materials. the procedure was identical to study with the exception of the dependent measure. instead of conspiracy ideation, we measured belief in fifteen specific conspiracies (e.g., a powerful and secretive group, known as the new world order, are planning to eventually rule the world through an autonomous world government, which would replace sovereign government), using the belief in conspiracy theories inventory (bcti), developed and later modified by swami and colleagues [ , ] . (we used the modified version) participants rated the conspiracy theories on a (completely false) to (completely true) scale, with higher scores indicating higher conspiracy theory beliefs. eight participants (all low control group) were excluded from the analysis, one because they self-assessed their data as unreliable and seven because they were not naïve to the research aims. a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = . . the neutral group scored numerically higher on conspiracy theory beliefs (m = . , sd = . , n = ) than the low control group (m = . , sd = . , n = ), but the difference was not significant t ( ) = . , p = . , f = . . ostensibly, then, the null effects in studies - do not appear to be due to the instrument used to measure conspiracy theory beliefs. although our four studies show little evidence for a main effect of control priming on conspiracy beliefs, it is entirely possible that additional factors moderate the effects of controlthat control motivates conspiracy beliefs in some circumstances but not others. in the final two studies, we explore two of the most plausible possibilities: the degree to which a conspiracy theory itself implies a controlling entity (study ); and the availability of other means of restoring control when it is threatened (study ). if people turn to conspiracy theories as a compensatory control mechanism, it may be that some theories are more effective at restoring perceptions of order than others. in particular, threats to control may only prompt belief in conspiracies involving a controlling entity (e.g., the theory that the u.s.s.r. started the "hippie" movement in the united states in order to undermine and weaken america's traditional values), because the belief that someone is in control is sufficient to satisfy the higher order need for structure and meaning. other theories in which a controlling entity is absent or only implicit (e.g., the theory that osama bin laden died prior to the / terrorist attacks but was used as a scapegoat) may not restore control, or do so to a lesser extent. consistent with this proposal, kay et al. [ ] found that participants whose control was threatened increased their belief in god, but only when god was described as controlling, rather than as a creator. thus, in study , we developed a new stimulus set to test the hypothesis that participants whose control was threatened would be inclined to vest more belief in specific conspiracy theories about controlling entities, but not in other conspiracy theories. participants. the participants were three hundred mturk workers ( males, females, m = . , sd = . ). the majority ( %) had an undergraduate degree, while % had a high school degree, % had a master's degree, % a doctoral degree and % did not have a degree. the mean age was . years (range to years). none had taken part in our previous studies. materials. the dependent measure was developed in several stages. first, an independent group of mturk participants (n = ) were asked to write five conspiracy theories that they had heard before, from which we identified unique exemplars. next, two research assistants were asked to code these theories on a -point scale anchored at ("the goal of this conspiracy is not control") and ("the goal of this conspiracy is control"). the intraclass correlation coefficient was r = . . the final rating comprised the average of the ratings of the two research assistants. next, a second group of mturk participants was asked to rate the conspiracy theories in terms of their prototypicality, using a seven point scale anchored at ("a bad fit" with their idea of a conspiracy theory) and (an "excellent fit" with their idea of a conspiracy theory). a third group of mturk participants was asked to rate the same conspiracies for likely veracity, using a nine point scale anchored at ("definitely false") and ("definitely true"). the intraclass correlation coefficient for prototypicality ratings was r = . and for the veracity r = . . the prototypicality and veracity scores for each conspiracy theory were obtained by averaging ratings, respectively across all participants. these extensive pretest data were used to create seven pairs of conspiracy theories that were equated on veracity (t ( ) = . , p = . , d = . ) and prototypicality (t ( ) = . , p = . , d = . ), but that each differed by at least scale points in terms of control (ms = . vs. . ). for the full list of the selected conspiracies, along with their control, prototypicality and veracity rating (obtained from the pre-test) see table , for the full list of all pretest conspiracies see the online supplementary materials. procedure. the procedure was identical to that of study , except for the use of two types of specific conspiracy theories, varying in the control they imply, and the addition of a high control group as additional comparison group. participants rated their agreement with the conspiracy statements, presented in a random order, on a -point scale ( = strongly disagree, = strongly agree). mixed among these statements was an attention check question (i.e., "as a response to this question select strongly agree."). nineteen participants were excluded from the analysis: nine (low control = , neutral = ) participants due to failures of attention and ten (low control = , high control = ) because they were not naïve to the research aims. a sensitivity analysis (α = . , β = . , correlation among repeated measures = . ) indicated that the study is able to detect a minimum effect size of f = . . a mixed anova with control manipulation (low control vs. neutral vs. high control) as between subject factor, and type of conspiracy theory (control related theory or non-control related theory) as a repeated measure, revealed no main effect of the manipulation f . crucially for our purposes, there was no conspiracy theory type x control manipulation interaction f ( , ) = . , p = . , f = . (the pattern of results was unchanged when we analyzed the low control vs. neutral groups only). we also analysed each pair separately. the interaction was not significant for any individual pair. in sum, we found no support for the hypothesis that control threat motivates belief in theories that hypothesize a controlling entity. in addition, participants rated the theories as more believable when they did not afford control, an unexpected result given that the two types of theories had been matched for believability in pretesting. to explore this discrepancy further, we compared our participants to the pretest sample on key demographic variables: age, gender, education and political ideology. the analyses indicated a marginal difference in the gender distribution, χ = . ( ), p = . ; there were a greater proportion of women in the main study compared to the pretest. in addition, women rated the control unrelated conspiracy theories as more believable than the control related, t( ) = - . , p< . , d = . . when we reran the repeated measures anova, with gender as a covariate, the main effect of conspiracy theory type was not present, f ( , ) = . , p = . , f = . and neither were the main effect of group, f ( , ) = . , p = . , f = . , nor the interaction effect f ( , ) = . , p = . , f = . . thus, the significant effect of conspiracy theory type was most likely due to the higher proportion of women in the main study. another plausible moderating factor determining the value of conspiracy theories for restoring threatened control, is the availability of alternative means of control. as noted above, while compensatory control processes do not require that sources of control be benevolent, people unsurprisingly favour sources of control that are not themselves threatening in other ways. furthermore, there is a plethora of alternative control and meaning-making systems that are proven substitutes for personal control, such as god [ ] , government institutions [ ] , scientific [ ] , and even more abstract beliefs such as in meritocracy [ ] and societal progress [ , ] . even if these alternatives are not all equally effective at establishing a sense of control, conspiracy theories would seem to be an option of last resort, given the essentially malevolent worldview they imply, and the social stigma that can accompany their endorsement [ ] . consequently, conspiracy theorizing may be a secondary strategy for restoring control, avoided when one has other means of affirming that the world is ordered [ ] . thus, in study , we tested the hypothesis that threats to control would increase belief in conspiracy theories to a greater degree (or only) when the effectiveness of a second salient source of control-the government [ ] -was called into question. participants. six hundred and five mturk volunteers participated ( male, female, "other"). the majority ( . %) had an undergraduate degree, . % had a high school degree, while . % had a graduate degree, . % an "other" degree and . % had no degree. the mean age was . years (sd = . age range . none had participated in our previous studies. materials and procedure. participants were randomly assigned to a low control or a neutral group, as described in previous studies, and, independently, to a "competent government" or "incompetent government" condition. in the former, participants read a passage (about which they expected to answer questions) describing the u.s. government's response, generally considered competent and effective [ ] , to hurricane irma which most severely struck florida, georgia and south carolina in ; in the latter, they read a similar passage about the government's response to hurricane katrina, generally considered incompetent and ineffective [ ] , which struck florida and louisiana in august . for example, hurricane irma's response was described as "timely, well managed, planned and coordinated. residents were ordered to a shelter of last resort with sufficient provision of food, water, security and sanitary conditions. no one was thirsty, exhausted or victim to violence." hurricane katrina's response was described as "delayed, mismanaged, unprepared and uncoordinated. residents were ordered to a shelter of last resort without provision of adequate food, water, security or sanitary conditions. several citizens died of thirst, exhaustion and violence, days after the storm had passed." (the full text of both scenarios appear in the appendix.) afterwards, consistent with the cover story, participants answered several multiple choice questions about the scenario (which were not analyzed). afterwards, all participants completed the conspiracy mentality scale, as described in study , including an attention check, followed by demographic questions and self-assessment of data quality. one hundred and four participants were excluded from the main analysis: due to failures of attention and due to failure to following instructions. this left a total of participants. a sensitivity analysis (α = . , β = . ) indicated that the study is able to detect a minimum effect size of f = . . table for descriptive statistics). thus, the study, again, provides no evidence that control priming influences conspiracy beliefs, regardless of whether an alternative source of control has been compromised. although studies - varied somewhat in methodology, their consistent inclusion of low control and neutral conditions permitted a meta-analysis, which maximizes both power and accuracy of our effect size estimates. because in study the low control manipulation did not lower perceived control compared to the neutral group we excluded study from the analysis. for studies (low control compared to neutral) and we looked at the effect at each level of the moderator. because the effect sizes in studies and were not independent we used the robumeta [ ] package in r, which takes the dependency into account, to conduct the meta-analysis. a test of heterogeneity suggested consistency between the studies, i = . the cumulative effect size did not suggest effect of condition, d = - . , % ci [- . , . ], p = . (fig ) . to investigate support for the null hypothesis relative to the alternative hypothesis that control priming influences conspiracy theory beliefs, we used a bayesian information criterion (bic), using the calculator by masson [ ] , based on wagenmakers [ ] . the analysis requires transformation of the sum of squares generated by an anova output and provides graded level of evidence for the null and alternative hypothesis. according to raftery [ ] , the strength of evidence for the posterior probability of a hypothesis given the data (p bic (h |d) within the range of . - . is "weak", between . - . is "positive", between . - . "strong" and above . "very strong". for those studies containing three groups we analysed the low and neutral (study and ) or low and high (study ). for studies and we looked at each level of the moderator. table . shows the posterior probability of the null hypothesis given the data, p bic (h |d), and the probability of the alternative hypothesis given the data, p bic (h |d). there is positive evidence for the null hypothesis in five out of the six studies. in study , there is only weak evidence for the null hypothesis, but the support for the alternative hypothesis is even lower (unsurprisingly, given that the effect is in the "wrong" direction). six studies, conducted online and using mturk participants, tested the hypothesis that lack of control motivates belief in conspiracies. while the hypothesis is plausible, there was in fact little support for it in the (small) literature on the subject. the current findings offer no further evidence, and some positive evidence for the null hypothesis, at least when the most common experimental manipulation of control is used with online samples. participants who were asked to recall instances in which they felt out of control were no more likely to endorse either general conspiracy beliefs, or specific conspiracy theories, relative to participants who recalled neutral memories, or nothing at all. plausible moderating variables-the extent to which conspiracies provide evidence of control (study ), and the effectiveness of an alternative means of control (study )-had no effect, and a meta-analysis of the studies determined a cumulative effect size not statistically different from zero. on the contrary, a bayesian analysis indicated that the null hypothesis (i.e., that there is no causal relationship between lack of control and conspiracy theory beliefs) should be retained. how to explain the inconsistency between previous positive results and our null findings? one explanation, of course, is that previous "positive" findings were simply type errors, which is not farfetched given social psychology's historical inattention to power [ , ] combined with publication bias. indeed, assuming the "actual" effect size of control threat on conspiracy beliefs is . (the average effect size reported in psychology research [ ] ), then previous studies reporting positive results may have included chance findings given their low [ ] or nyhan & zeitzoff [ ] , have higher power, . and . , respectively. another possibility, however, is that some previous studies measured constructs other than "pure" conspiracy ideation (e.g., pattern perception, paranoid beliefs, corruption beliefs), which are susceptible to control threats. indeed, studies that examine more typical conspiracy theories and have larger samples tend to report null results. for example, hart and graether [ ] , found no effects of control on the generic conspiracist beliefs scale (another psychometrically validated measure of conspiracy beliefs, with items such as, "experiments involving new drugs or technologies are routinely carried out on the public without their knowledge or consent"). similarly, nyhan and zeitzoff [ ] found no effect of control priming on middle eastern and north african participants' conspiracy beliefs, using a list of conspiracy theories consisting of items such as "jewish leaders are secretly conspiring to achieve world domination." it is also worth repeating that the current results were obtained entirely with online samples, while most studies in this area (including the majority of those with positive effects of control) have been conducted in the laboratory (typically with student samples). in principle, online manipulations of control could be weaker than those in the laboratory, accounting for the lack of support for the control hypothesis. as an empirical matter, however, there is no evidence that effects in this paradigm depend on sample type [ ] . in a recent meta-analysis of experimental manipulations of control on conspiracy beliefs [ ] conducted on effect sizes across studies (including those reported here), we found no moderating effect of sample type (mturk vs. students vs. other). thus, although it is important to continue to examine the role of control threats in diverse samples and contexts, the current data, despite being collected online, nevertheless challenge the hypothesis that such threats account for conspiracy beliefs in any significant way. although there was no evidence in the current studies for a causal effect of control on conspiracy beliefs, studies and provided evidence for a correlational relationship. such correlational findings are consistent with previous research [ , , , , ] , suggesting that chronic, rather than acute, feelings of low control are related to higher conspiracy theory beliefs. while longitudinal studies may help shed light on the effects of chronic lack of control on conspiracy beliefs, it is also possible that the link is spurious. there are any number of covariates of both lack of control and conspiracy theory belief, including stress [ , ] , selfesteem [ , ] , anxiety [ , ] or uncertainty [ ] , that could account for their association. also, the correlation may be due to conspiracy beliefs' shared variance with paranoia (see for example imhoff & lamberty, [ ] ). alternatively, the causality may run in the opposite direction: conspiracy theories themselves may reduce feelings of control. jolley and douglas [ ] , for example, found that participants exposed to conspiracy theories about vaccines reported greater feelings of powerlessness compared to participants exposed to anti-conspiracy material, suggesting that conspiracy beliefs threaten rather than satiate one's sense of control. furthermore, it is not clear what type of correlational relationship should be taken as evidence for satiation; if conspiracy theories compensate for compromised personal control, should the relationship between control and conspiracy beliefs in cross-sectional studies be positive or negative? thus, correlational evidence in the absence of experimental evidence is not a strong foundation for claiming that lack of control is a precursor to conspiracy beliefs. even if our present null results are confirmed in subsequent research, it is important to acknowledge their limitations. priming is only one method of threatening (or bolstering) a sense of personal control, and while we found no evidence for changes in conspiracy beliefs, more potent or qualitatively distinct experimental manipulations could conceivably produce effects that we were unable to detect here. furthermore, we could not assess the effects of chronic lack of control: it may be that prolonged periods of control deprivation do indeed produce reliance on other, even malevolent sources of control, including conspiracy theories. another unexplored area is whether lack of control in a specific domain will lead to belief in conspiracy theory in that domain. for example, people diagnosed with hiv or covid- might be particularly inclined to believe in conspiracy theories involving those viruses, rather than conspiracy theories in general. indeed, in a recent naturalistic study [ ] , we found a domain-specific link between threat to control and conspiracy beliefs. for example, after a series of tornados, those who experienced such natural disasters were more likely to endorse weather-related conspiracy beliefs than they were other types of conspiracy beliefs. moreover, the strength of these endorsements subsided over time (i.e., three months after tornado season). such findings seem to suggest that the link between perceived control and conspiracy beliefs is both nuanced and transitory, and that there is no "one size fits all" explanation for lack of control and conspiracy-theory endorsement in general. finally, we relied exclusively on laboratory manipulations of control (or lack thereof). while such empirical techniques are widely used in the literature, the effectiveness of the "recall task" in threatening participants' sense of control has been questioned [ ] , and its effects were weak in our studies. "real life" events, such as natural disasters, terrorist attacks, and pandemics, may produce stronger threats to personal control (and indeed, evidence suggests that traumatic events are followed by an increase in conspiracy discourse [ ] ). findings from our own naturalistic study seems to confirm this. however, such naturalistic "manipulations" may confound perceptions of control with other variables, such as threats to identity, uncertainty about the future, and stress. thus, any naturalistic observations need to be accompanied by rigorous experimental research to triangulate the mechanism(s) responsible for any changes in conspiracy beliefs. on their own, both empirical approaches are problematic, in that experimental manipulations of control may be too "weak" to evoke conspiracy-belief endorsements, and perceived loss of control under naturally occurring conditions are confounded with other variables. the conspiracy mentality scale: distinguishing between irrational and rational suspicion anxious attachment and belief in conspiracy theories conspiracist ideation in britain and austria: evidence of a monological belief system and associations between individual psychological differences and real-world and fictitious conspiracy theories they will not control us': ingroup positivity and belief in intergroup conspiracies the effect of intergroup threat and social identity salience on the belief in conspiracy theories over terrorism in indonesia: collective angst as a mediator the role of social identification, intergroup threat, and out-group derogation in explaining belief in conspiracy theory about terrorism in indonesia belief in conspiracy theories and susceptibility to the conjunction fallacy intention seekers: conspiracist ideation and biased attributions of intentionality someone is pulling the strings: hypersensitive agency detection and belief in conspiracy theories suspicious minds here's why people believe in conspiracy theories producer) & alpert d. 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anxiety and task interference the effect of high-anxiety situations on conspiracy thinking: research and reviews research and reviews belief in conspiracy theories: the influence of uncertainty and perceived morality how paranoid are conspiracy believers? toward a more fine-grained understanding of the connect and disconnect between paranoia and belief in conspiracy theories the effects of anti-vaccine conspiracy theories on vaccination intentions perceived lack of control and conspiracy theory beliefs in the wake of political strife and natural disaster. under review conspiracy theories as part of history: the role of societal crisis situations conceptualization: ana stojanov, jamin halberstadt. key: cord- -wawui fd authors: tulchinsky, theodore h.; varavikova, elena a. title: communicable diseases date: - - journal: the new public health doi: . /b - - / - sha: doc_id: cord_uid: wawui fd publisher summary in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. public health has addressed the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. important for all health providers and public health personnel so as to be able to cope with the scale of these problems and to absorb new technologies as they emerge from scientific advances and experience, and their successful application. lived. it was to be observed, indeed, that it did not come straight on toward us; for the city, that is to say within the walls, was indifferently healthy still; nor was it got over the water into southwark; for though there died that week , of all distempers, whereof it might be supposed above died of the plague, yet there was but in southwark, lambeth parish included; whereas in the parishes of st. giles the agent-host-environment triad, discussed in chapter , is fundamental to the success of understanding transmission of infectious diseases and their control, including those well known, those changing their patterns, and those newly emerging or escaping current methods of control. infection occurs when the organism successfully invades the host body, where it multiplies and produces an illness. a host is a person or other living animal, including birds and arthropods, who provides a place for growth and sustenance to an infectious agent under natural, as opposed to experimental, conditions. some organisms, such as protozoa or helminths, may pass successive stages of their life cycle in different hosts, but the primary or definitive host is the one in which the organism passes its sexual stage. the secondary or intermediate host is where the parasite passes the larval or asexual stage. a transport host is a carrier in which the organism remains alive, but does not develop. an agent of an infectious disease is necessary, but not always sufficient to cause a disease or disorder. the infective dose is the quantity of the organism needed to cause clinical disease. a disease may have a single agent as a cause, or it may occur as a result of the agent in company with contributory factors, whose presence is also essential for the development of the disease. a disease may be present in an infected person in a dormant form such as tuberculosis, or a subclinical form, such as poliomyelitis or hiv. the virulence or pathogenicity of an infective agent is the capacity of an infectious agent to enter the host, replicate, damage tissue, and cause disease in an exposed and susceptible host. virulence is indicated by the severity of clinical disease and case fatality rates. the environment provides a reservoir for the organism, and the mode of transmission, by which the organism reaches a new host. the reservoir is the natural habitat where an infectious agent lives and multiplies, from which it can be transmitted directly or indirectly to a new host. the reservoir refers to the natural habitat of the organism, which may be in people, animals, arthropods, plants, soil, or substances in which an organism normally lives and multiplies, and on which it depends for survival or in which it survives in a dormant form. contacts are persons or animals who have been in association with an infected person, animal, or contaminated inanimate object, or environment that might provide an opportunity for acquiring the infective agent. persons or animals that harbor a specific infectious agent, often in the absence of discernible clinical disease, and who serve as a source of infection or contamination of food, water, or other materials, are carriers. a carrier may have an inapparent infection (a healthy cartier) or may be in the incubation or convalescent stage of the infection. communicable diseases may be classified by a variety of methods: by organism, by mode of transmission, by methods of prevention (e.g., vaccine preventable, vector controllable), or by major organism classification, that is, viral, bacterial, and parasitic disease. a virus is a nucleic acid molecule (rna or dna) encapsulated in a protein coat or capsid. the virus is not a complete cell and can only replicate inside a complete cell. the capsid may have a protective envelope of a lipid containing membrane. the capsid and membrane facilitate attachment and penetration of a host cell. inside the host cell, the nucleic molecule may cause the cell's chromosomes to be changed in its own genetic material or so that there is cellular manufacture and virus replication. viroids are smaller rna structures without capsids which can cause plant disease. prions are recently discovered (stanley prusiner, nobel prize, ) variants of viruses or viroids which are the infective agents cause of scrapie in sheep, and similar degenerative central nervous system diseases in cattle and in man (mad cow disease or creutzfeld-jakob disease in humans). bacteria are unicellular organisms that reproduce sexually or asexually, grow on cell-free media, and can exist in an environment with oxygen (aerobic) or in one lacking oxygen (anaerobic). some may enter a dormant state and form spores where they are protected from the environment and may remain viable for years. bacteria include a nucleus of chromosomal dna material within a membrane surrounded by cytoplasm, itself enclosed by the cellular membrane. bacteria are often characterized by their coloration under gram's stain, as gram-negative or gram-positive, as well as by their microscopic morphology, colony patterns on growth media, by the diseases they may cause, as well as by antibody and molecular (dna) marking techniques. bacteria include both indigenous flora (normal resident) bacteria and pathogenic (disease causing) bacteria. pathogenic bacteria cause disease by invading, overcoming natural or acquired resistance, and multiplying in the body. bacteria may produce a toxin or poison that can affect a body site distant from where the bacterial replication occurs, such as in tetanus. bacteria may also initiate an excessive immune response, producing damage to other body tissues away from the site of infection, e.g;, acute rheumatic fever and glomerulonephritis. parasitology studies protozoa, helminths, and arthropods that live within, on, or at the expense of a host. these include oxygen-producing, flagellate, unicellular organisms such as giardia and trichomonas, and amoebas such as entamoeba important in enteric and gynecologic disorders. sporozoa are parasites with complex life cycles in different hosts, such as cryptosporidium or malarial parasites. parasitic disease usually refers to infestation, with fungi, molds, and yeasts that can affect humans. helminths are worms that infest humans especially in poor sanitation and tropical areas. transmission of diseases is by the spread of an infectious agent from a source or reservoir to a person (table . ). direct transmission from one host to another occurs during touching, biting, kissing, sexual intercourse, and projection via droplets, as in sneezing, coughing, or spitting, or by entry through the skin. indirect transmission includes via aerosols of long-lasting suspended particles in air, fecal-oral transmission such as food and waterborne as well as by poor hygenic conditions with inanimate materials, such as soiled clothes, handkerchiefs, toys, or other objects. vector-borne diseases are transmitted via crawling or flying insects, in some cases with multiplication, and development of the organism in the vector, as in malaria. the subsequent transmission to humans is by injection of salivary gland fluid during biting, e.g., congenital syphilis, or by deposition of feces, urine or other material capable of penetrating the skin through a bite wound or other trauma. transmission may occur with insects as a transport mechanism, as in salmonella on the legs of a housefly. airborne transmission occurs inderectly via infective organisms in small aerosols that may remain suspended for long periods of time and which easily enter the respiratory tract. small particles of dust may spread organisms from soil, clothing, or bedding. vertical transmission occurs from one generation to another, or from one stage of the insect life cycle to another stage. maternal-infant transmission occurs during pregnancy (transplacental), delivery, as in gonorrhoea, breast-feeding, e.g., hiv, with transfer of infectious agents from mother to fetus or newborn. resistance to infectious diseases is related to many host and environmental factors, including age, sex, pregnancy, nutrition, trauma, fatigue, living and socioeconomic conditions, and emotional status. good nutritional status has a protective effect against the results of an infection. vitamin a supplements reduce complication rates of measles and enteric infections. tuberculosis may be present in an individual whose resistance is sufficient to prevent clinical disease, but the infected person is a cartier of an organism which can be transmitted to another or cause clinical disease if the person's susceptibility is reduced. immunity is resistance to infection resulting from presence of antibodies or cells that specifically act on the microorganism associated with a specific disease or toxin. immunity to a specific organism can be acquired by having the disease, that is, natural immunity, or by immunization, active or passive, or by protection box . vaccines and prevention "the greeks had two gods of health, aesculapius and hygeia, therapy and prevention, respectively. medicine in the twentieth century retains those two concepts, and vaccination is a powerful means of prevention. what follows is information on the vaccines that together with sanitation, make modem society possible, and that if wisely used will continue to bestow on mankind the gift of prevention, which according to proverb is worth far more than cure." source: plotkin, s. a., mortimer, e. a. . vaccines. second edition. philadelphia: wb saunders (with permission). infectious agent: a pathogenic organism (e.g., virus, bacteria, rickettsia, fungus, protozoa, or helminth) capable of producing infection or an infectious disease. infection: the process of entry, development, and proliferation of an infectious agent in the body tissue of a living organism (human, animal, or plant) overcoming body defense mechanisms, resulting in an inapparent or clinically manifest disease. antigen: a substance (e.g., protein, polysaccharide) capable of inducing specific response mechanisms in the body. an antigen may be introduced into the body by invasion of an infectious agent, by immunization, inhalation, ingestion, or through the skin, wounds, or via transplantation. antibody: a protein molecule formed by the body in response to a foreign substance (an antigen) or acquired by passive transfer. antibodies bind to the specific antigen that elicits its production, causing the infective agent to be susceptible to immune defense mechanisms against infections e.g., humoral and cellular. immunoglobulins: antibodies that meet different types of antigenic challenges. they are present in blood or other body fluids, and can cross from a mother to fetus in utero, providing protection during part of the first year of life. there are five major classes (igg, igm, iga, igd, and ige) and subclasses based on molecular weight. anfisera or antitoxin: materials prepared in animals for use in passive immunization against infection or toxins. source: jawetz, melrick, and adelberg, medical microbiology, . through elimination of circulation of the organism in the community. immunity may be by antibodies produced by the host body or transferred from externally produced antibodies. the body also reacts to infective antigens by cellular responses, including those that directly defend against invading organisms and other cells which produce antibodies. the immune response is the resistance of a body to specific infectious organisms or their toxins provided by a complex interaction of antibodies and cells including a. b cells (bone marrow and spleen) produce antibodies which circulate in the blood, i.e., humoral immunity; b. t cell-mediated immunity is provided by sensitization of lymphocytes of thymus origin to mature into cytotoxic cells capable of destroying virusinfected or foreign cells; c. complement, a humoral response which causes lysis of foreign cells; d. phagocytosis, a cellular mechanism which ingests foreign microorganisms (macrophages and leukocytes). surveillance of disease is the continuous scrutiny of all aspects of occurrence and spread of disease pertinent to effective control of that disease. maintaining ongoing surveillance is one of the basic duties of a public health system, and is vital to the control of communicable disease, providing the essential data for tracking of disease, planning interventions, and responding to future disease challenges. surveillance of infectious disease incidence relies on reports of notifiable diseases by physicians, supplemented by individual and summary reports of public health laboratories. such a system must concern itself with the completeness and quality of reporting and potential errors and artifacts. quality is maintained by seeking clinical and laboratory support to confirm first reports. completeness, rapidity, and quality of reporting by physicians and laboratories should be emphasized in undergraduate and postgraduate medical education. enforcement of legal sanctions may be needed where standards are not met. surveillance of infectious diseases includes the following: . morbidity reports from clinics to public health offices; . mortality reports from attending doctors to vital records; . reports from selected sentinel centers; . special field investigations of epidemics or individual cases; . laboratory monitoring of infectious agents in population samples; . data on supply, use, and side effects of vaccines, toxoids, immune globulins; . data on vector control activities such as insecticides use; . immunity levels in samples of the population at risk; . review of current literature on the disease; . epidemiologic and clinical reports from other jurisdictions. epidemiologic monitoring based on individual and aggregated reports of infectious diseases provide data vital to planning interventions at the community level or for the individually exposed patient and his contacts, along with other information sources such as hospital discharge data and monitoring of sentinel centers. these may be specific medical or community sites that are representative of the population and are able to provide good levels of reporting to monitor an area or population group. a sentinel center can be a pediatric practice site, a hospital emergency room, or other location which will provide a "finger on the pulse" to assess the degree and kind of morbidity occurring in the community. it can also include monitoring in a location previously known for disease transmission, such as hong kong in relation to influenza. epidemiologic analysis provided by government public health agencies should be published weekly, monthly, and annually and distributed to a wide audience of public health and health-related professionals throughout the country. feedback to those in the field on whose initial reports the data are based is vital in order to promote involvement and improved quality of data, as well as to allow evaluation of the local situation in comparison to other areas. in a federal system of government, national agencies report regularly on all state or provincial health patterns. state or provincial health authorities provide data to the counties and cities in their jurisdictions. such data should also be readily available to researchers in other government agencies, universities, and other academic settings for further research and analysis both on internet and hard-copy publications. notifiable diseases are those which a physician is legally required to report to state or local public health officials, by reason of their contagiousness, severity, frequency, or other public health importance (table . ). public health laboratory services provide validation of clinical and epidemiologic reports. they also pro- vide day-to-day supervision of public health conditions, and can monitor communicable disease and vaccine efficacy and coverage. in addition, they support standards of clinical laboratories in biochemistry, microbiology, and genetic screening. nosocomial or hospital-acquired infections constitute a major health hazard associated with care in institutions. in the united states, they occur in - % of hospital admissions and are the cause of lengthening of hospital stay and an estimated , deaths per year. in developing countries, nosocomial infection rates may occur in up to % of hospitalizations. this category of infectious disease most commonly includes infections of the urinary tract, surgical wounds, lower respiratory tract (pneumonias), and blood poisoning or septicemias. in the united states, up to % of hospital-acquired infections are caused by multidrug resistant organisms. staphylococcus infections resistant to many current antibiotics, for example, methicillin and vancomycin, are a notable cause of prolongation of hospitalization or even death. the increasing number of immunodeficient patients has increased the importance of prevention of nosocomial infections. where standards of infection control are lacking, in both developed and developing countries, hospital staff are vulnerable to serious infection. in developing countries, deadly new viruses, such as ebola and marburg viruses mainly affect nursing, medical, and other staff as secondary cases. surveillance and control measures are important elements of hospital management. hospital epidemiologists and infection control staff are part of modem hospital staffing. the cost to the health system of nosocomial infections is a major consideration in planning health budgets. reducing the risk of acquiring such infections in hospital justifies substantial expenditures for hospital epidemiology and infection control activities. with diagnostic related group payment for hospital care (by diagnosis rather than by days of stay) the good manager has a major incentive to ensure that the risk of nosocomial infections is minimized, since they can greatly prolong hospital stays, raising patient dissatisfaction and health care costs. an endemic disease is the constant usual presence of a disease or infectious agent in a given geographic area or population group. hyperendemic is a state of persistence of high levels of incidence of the disease. holoendemic means that the disease appears early in life and affects most of the population, as in malaria or hepatitis a and b in some regions. an epidemic is the occurrence in a community or region of a number of cases of an illness in excess of the usual or expected number of cases. the number of cases constituting an epidemic varies with the disease, and factors such as previous epidemiological patterns of the disease, time and place of the occurrence, and the population involved must be taken into account. a single case of a disease long absent from an area, such as polio, constitutes an epidemic, and therefore a public health emergency because a clinical case may represent a hundred carriers with nonparalytic or subclinical poliomyelitis. in the s, two to three or more cases of measles linked in time and place may be considered sufficient evidence of transmission and presumed to be an epidemic. a pandemic is occurrence of a disease over a very wide area, crossing international boundaries, affecting a large proportion of the population. each epidemic should be regarded as a unique natural experiment. the investigation of an epidemic requires preparation and field investigation in conjunction with local health and other relevant authorities. verification of cases and the scope of the epidemic will require case definition and laboratory confirmation. tabulation of known cases according to time, place, and person are important for immediate control measures and formulation of the hypothesis as to the nature of the epidemic. an epidemic curve is a graphic plotting of the distribution of cases by the time of onset or reporting, which gives a picture of the timing, spread, and extent of the disease from the time of the initial index cases and the secondary spread. epidemic investigation requires a series of steps. this starts with confirmation of the initial report and preliminary investigation, defining who is affected, determining the nature of the illness and confirming the clinical diagnosis, and recording when and where the first (index) and follow-up (secondary) cases occurred, and how the disease was transmitted. samples are taken from index case patients (e.g., blood, feces, throat swabs) as well as from possible vectors (e.g., food, water, sewage, environment). a working hypothesis is established based on the first findings, taking into account all plausible explanations. the epidemic pattern is studied, establishing common source or risk factors, such as food, water, contact, environment, and drawing a time line of cases to define the epidemic curve. how many are ill (the numerator) and what is the population at risk (the denominator) establish the attack rate, namely, the percentage of sick among those exposed to the common factor. what is a reasonable explanation of the occurrence; is there a previous pattern, with the present episode a recurrence or new event? consultation with colleagues and the literature helps to establish both a biological and epidemiologic plausibility. what steps are needed to prevent spread and recurrence of the disease? coordination with relevant health and other officials and providers is required to establish surveillance and control systems, document and distribute reports, and respond to the public's fight to know. the first reports of excess cases may come from a medical clinic or hospital. the initial (sentinel or index) cases provide the first clues that may point to a common source. investigation of an epidemic is designed to quickly elucidate the cause and points of potential intervention to stop its continuation. this requires skilled investigation and interpretation. epidemiologic investigations have defined many public health problems. rubella syndrome, legionnaire's disease, aids, and lyme and hantavirus diseases were first identified clinically when unusually large numbers of cases appeared with common features. the suspicions that were raised led to a search for causes and the identification of control methods. a working hypothesis of the nature of an epidemic is developed based on the initial assessment, the type of presentation, the condition involved, and previous local, regional, national, and international experience. the hypothesis provides the basis for further investigation, control measures, and planning additional clinical and laboratory studies. surveillance will then monitor the effectiveness of control measures. communication of findings to local, regional, national, and international health reporting systems is important for sharing the knowledge with other potential support groups or other areas where similar epidemics may occur. the centers for disease control and prevention (cdc), originally organized in as the office for malaria control in war areas, is part of the u.s. public health service. as of , the cdc had a budget of $ . billion, and employees include epidemiologists, microbiologists, and many other professionals. the cdc includes national centers for environmental health and injury control, chronic disease prevention and health promotion, infectious diseases, prevention services, health statistics, occupational safety and health, and international health. the epidemic intelligence service (eis) of the cdc in the united states is an excellent model for the organization of the national control of communicable diseases. young clinicians are trained to carry out epidemiologic investigations as part of training to become public health professionals. eis officers are assigned to state health departments, other public health units, and research centers as part of their training, carrying out epidemic investigation and special tasks in disease control. the cdc, in cooperation with the who, has developed and offers free of charge, a personal computer program to support field epidemiology, including epidemic investigations (epi-info), which can be accessed and down-loaded from the worldwide web. this program should be adopted widely in order to improve field investigations, to encourage reporting in real time, and to develop high standards in this discipline. cdc's morbidity and mortality weekly report (mmwr) is a weekly publication of the cdc's epidemiologic data, also available free on the internet. it includes special summaries of reportable infectious diseases as well as noncom- although an infectious disease is an event affecting an individual, it is communicable to others, and therefore its control requires both individual and community measures of protection. control of the disease is a reduction in its incidence, prevalence, morbidity, and mortality. elimination of a disease in a specified geographic area may be achieved as a result of intervention programs such as individual protection against tetanus; elimination of infections such as measles requires stoppage of circulation of the organism. eradication is success in reduction to zero of incidence of the disease and presence in nature of the organism, such as with smallpox. extinction means that a specific organism no longer exists in nature or in laboratories. public health applies a wide variety of tools for the prevention of infectious diseases and their transmission. it includes activities ranging from filtration and disinfection of community drinking water to environmental vector control, pasteurization of milk, and immunization programs (see table . ). no less important are organized programs to promote self protection, case finding, and effective treatment of infections to stop their spread to other susceptible persons (e.g., hiv, sexually transmitted diseases, tuberculosis, malaria). planning measures to control and eradicate specific communicable diseases is one of the principal activities of public health and remains so for the twenty-first century. treating an infection once it has occurred is vital to the control of a communicable disease. each person infected may become a vector and continue the chain of transmission. successful treatment of the infected person reduces the potential for an uninfected contact person to acquire the infection. bacteriostatic agents or drugs such as sulfonamides inhibit growth or stop replication of the organism, allowing normal body defenses to overcome the organism. bacteriocidal drugs such as penicillin act to kill pathogenic organisms. traditional medical emphasis on single antibiotics has changed to use of multiple drug combinations for tuberculosis and more recently for hospital-acquired infections. antibiotics have made enormous contributions to clinical medicine and public health. however, pathogenic organisms are able to adapt or mutate and develop resistance to antibiotics, resulting in drug resistance. wide-scale use of antibiotics has led to increasing incidence of resistant organisms. multidrug resistance constitutes one of the major public health challenges at the end of the twentieth century. antiviral agents (e.g., ribovarin) are important additions to medical treatment potential, as are "cocktails" of antiviral agents for management of hiv infection. antibiotic use is a health problem requiting attention of clinicians and their teachers as well as the public health community and health care managers, representing the interaction of health issues across the entire spectrum of services. organized public health services are responsible for advocating legislation and for regulating and monitoring programs to prevent infectious disease occurrence and/or spread. they function to educate the population in measures to reduce or prevent the spread of disease. health promotion is one of the most essential instruments of infectious disease control. it promotes compliance and community support of preventive measures. these include personal hygiene and safe handling of water, milk, and food supplies. in sexually transmitted diseases, health education is the major method of prevention. each of the infectious diseases or groups of infectious diseases have one or more preventive or control approaches (table . ). these may involve the coordinated intervention of different disciplines and modalities, including epidemiologic monitoring, laboratory confirmation, environmental measures, immunization, and health education. this requires teamwork and organized collaboration. very great progress has been made in infectious disease control by clinical, public health, and societal means since in the industrialized countries and since the s in the developing world. this is attributable to a variety of factors, including organized public health services; the rapid development and wide use of new and improved vaccines and antibiotics; better access to health care; and improved sanitation, living conditions, and nutrition. triumphs have been achieved in the eradication of smallpox and in the increasing control of other vaccine-preventable diseases. however, there remain serious problems with tb, stds, malaria, and new infections such as hiv, and an increase in multiple drug-resistant organisms. vaccines are one of the most important tools of public health in the control of infectious diseases, especially for child health. vaccine-preventable diseases ta b l e . annual incidence of selected vaccine-preventable infectious diseases in rates per , population selected years, united states, - disease the body responds to invasion of disease-causing organisms by antigenantibody reactions and cellular responses. together, these act to restrain or destroy the disease-causing potential. strengthening this defense mechanism through im-box . definitions of immunizing agents and processes vaccines: a suspension of live or killed microorganisms or antigenic portion of those agents presented to a potential host to induce immunity to prevent the specific disease caused by that organism. preparation of vaccines may be from: a. live attenuated organisms which have been passed repeatedly in tissue culture or chick embryos so that they have lost their capacity to cause disease but retain an ability to induce antibody response, such as polio-sabin, measles, rubella, mumps, yellow fever, bcg, typhoid, and plague. b. inactivated or killed organisms which have been killed by heat or chemicals but retain an ability to induce antibody response; they are generally safe but less efficacious than live vaccines and require multiple doses, such as polio-salk, influenza, rabies, and japanese encephalitis. c. cellular fractions usually of a polysaccharide fraction of the cell wall of a disease-causing organisms, such as pneumococcal pneumonia or meningococcal meningitis. d. recombinant vaccines produced by recombinant dna methods in which specific dna sequences are inserted by molecular engineering techniques, such as dna sequences spliced to vaccinia virus grown in cell culture to produce influenza and hepatitis b vaccines. toxoids or antisera: modified toxins are made nontoxic to stimulate formation of an antitoxin, such as tetanus, diphtheria, botulism, gas gangrene, and snake and scorpion venom. immune globulin: an antibody-containing solution derived from immunized animals or human blood plasma, used primarily for short-term passive immunization, e.g., rabies, for immunocompromised persons. antitoxin: an antibody derived from serum of animals after stimulation with specific antigens and used to provide passive immunity, e.g., tetanus. munization is one of the outstanding achievements of public health, as treatment of infectious diseases by antimicrobials is a major element of clinical medicine. immunization (vaccination) is a process used to increase host resistance to specific microorganisms to prevent them from causing disease. it induces primary and secondary responses in the human or animal body: a. primary response occurs on first exposure to an antigen. after a lag or latent period of - days (depending on the antigen) specific antibodies appear in the blood. antibody production ceases after several weeks but memory cells that can recognize the antigen and respond to it remain ready to respond to a further challenge by the same antigen. b. secondary (booster) response is the response to a second and subsequent exposure to an antigen. the lag period is shorter than the primary response, the peak is higher and lasts longer. the antibodies produced have a higher affinity for the antigen, and a much smaller dose of the antigen is required to initiate a response. c. immunologic memory exists even when circulating antibodies are insufficient to protect against the antigen. when the body is exposed to the same antigen again, it responds by rapidly producing high levels of antibody to destroy the antigen before it can replicate and cause disease. immunization protects susceptible individuals from communicable disease by administration of a living modified agent, or subunit of the agent, a suspension of killed organisms or an inactivated toxin (see table . ) to stimulate development of antibodies to that agent. in disease control, individual immunity may also protect another individual. herd immunity occurs when sufficient persons are protected (naturally or by immunization) against a specific infectious disease reducing circulation of the organism, thereby lowering the chance of an unprotected person to become infected. each pathogen has different characteristics of infectivity, and therefore different levels of herd immunity are required to protect the nonimmune individual. the critical proportion of a population that must be immunized in order to interrupt local circulation of the organism varies from disease to disease. eradication of smallpox was achieved with approximately % world coverage, followed by concentration on new case findings and immunization of contacts and surrounding communities. for highly infectious diseases, such as measles, immunization coverage of over % is needed to achieve local eradication. immunization coverage in a community must be monitored in order to gauge the extent of protection and need for program modification to achieve targets of disease control. immunization coverage is expressed as a proportion in which the numerator is the number of persons in the target group immunized at a specific age, and the denominator is the number of persons in the target cohort who should have been immunized according to the accepted standard: vaccine coverage = no. persons immunized in specific age group • no. persons in the age group during that year immunization coverage in the united states is regularly monitered by the national immunization survey by a household survey in all states, as well as selected urban areas considered to be at high risk for undervaccination. an initial telephone survey is followed by confirmation, where possible, from documentation from the parents or health care providers. the survey for july -june examined children born between august and november (i.e., aged - months, median age months). the results show improving coverage, with % having received three or more doses of dpt (diphtheria, pertussis, and tetanus), % with three or more doses of opv (oral polio vaccine), % with three or more doses of haemophilus influenzae, type b (hib), but only % with three or more doses of hepatitis b. however, only % had received all recommended vaccines at the recommended ages. eases that still cause millions of deaths globally each year. other important infectious diseases are still not subject to vaccine control because of difficulties in their development. in some cases, a microorganism can mutate with changes. viruses can undergo antigenic shifts in the molecular structure in the organism, producing completely new subtypes of the organism. hosts previously exposed to other strains may have little or no immunity to the new strains. antigenic drift refers to relatively minor antigenic changes which occur in viruses. this is responsible for frequent epidemics. antigenic shift is believed to explain the occurrence of new strains of influenza virus necessitating, for example, annual reformulation of the influenza vaccine associated with large scale epidemics and pandemics. new variants of poliovirus strains are similar enough to the three main types so that immunity to one strain is carded over to the new strain. molecular epidemiology is a powerful new technique used to specify the geographic origin of organisms such as poliomyelitis and measles viruses, permiting tracking of the source of the virus and epidemic. combinations of more than one vaccine is now common practice with a trend to enlarging the cocktail of vaccines in order to minimize the number of injections, and visits required. this reduces the number of visits to carry out routine immunization saving staff time and costs, as well as increasing compliance. there are virtually no contraindications to use of multiple antigens simultaneously. examples of vaccine cocktails include dpt (diphtheria, pertussis, and tetanus) in combination with haemophilus influenzae b, poliomyelitis, and varicella, or mmr (measles, mumps, and rubella) vaccines. interventions in the form of effective vaccines save millions of lives each year and contribute to improved health of countless children and adults throughout the world. vaccination is now accepted as one of the most cost-effective health interventions currently available. continuous policy review is needed regarding allocation of adequate resources, logistical organization, and continued scientific effort to seek effective, safe, and inexpensive vaccines for other important diseases such as malaria and hiv. new technology of recombinant vaccines, such as that of hepatitis b, holds promise for important vaccine breakthroughs in the decades ahead. internationally, much progress was made in the s in the control of vaccinepreventable diseases. at the end of the s, fewer than % of the world's children were being immunized. who, unicef, and other international organizations mobilized to promote an expanded programme on immunization (epi) with a target of reaching % coverage by . immunization coverage increased in the developing countries, preventing some million child deaths annually. bacillus calmette-gu rin (bcg) coverage rose from to %; poliomyelitis with opv (three doses) from to %, and tetanus toxoid for pregnant women from to %. since , there has been a decline in coverage in some parts of the world, mainly in sub-saharan africa. the challenge remains to achieve control or eradication of vaccine-preventable diseases, thus saving millions of more lives. part of the hfa stresses the epi approach, which includes immunization against diphtheria, pertussis, tetanus, po-liomyelitis, measles, and tuberculosis. an extended form of this is the epi plus program which combines epi with immunization against hepatitis b and yellow fever and, where appropriate, supplementation with vitamin a and iodine. the success in international eradication of smallpox is now being followed by a campaign to eradicate poliomyelitis and other important infectious diseases. diphtheria. diphtheria is an acute bacterial disease of the tonsils, nasopharynx, and larynx caused by the organism corynebacterium diphtheriae. it occurs in colder months in temperate climates where the organism is present in human hosts and is spread by contact with patients or carriers. it has an incubation period of - days. in the past, this was primarily an infection of children and was a major contributor to child mortality in the prevaccine and preantibiotic eras. diphtheria has been virtually eliminated in countries with well-established immunization programs. in the s, an outbreak of diphtheria occurred in the countries of the former soviet union among people over age . it reached epidemic proportions in the s, with , cases ( - ) with deaths in in russia alone. this indicates a failure of the vaccination program in several respects: it used only three doses of dpt in infancy; no boosters were given at school age or subsequently; the efficacy of diphtheria vaccine may have been low, and coverage was below %. efforts to control the present epidemic include mass vaccination campaigns for persons over years of age with a single dose of dt (diphtheria and tetanus) and increasing coverage of routine dpt vaccines to four doses by age years. the epidemic and its control measures have led to improved coverage with dt for those over years, and % coverage among children aged - months. who recommends three doses of dpt in the first year of life and a booster at school entry. this is considered by many to be insufficient to produce long-lasting immunity. the united states and other industrialized countries use a four-dose schedule and recommend periodic boosters for adults with dt. pertussis. pertussis is an acute bacterial disease of the respiratory tract caused by the bacillus bordetella pertussis. after an initial coldlike (catarrhal) stage, the patient develops a severe cough which comes in spasms (paroxysms). the disease can last - months. the paroxysms can become violent and may be followed by a characteristic crowing or high pitched inspiratory whooping sound, followed by expulsion of a tenacious clear sputum, often followed by vomiting. in poorly immunized populations and those with malnutrition, pneumonia often follows and death is common. pertussis declined dramatically in the industrialized countries as a result of widespread coverage with dpt. however, because the pertussis component of the vaccine caused some reactions, many physicians avoided its use, using dt alone. during the s in the united kingdom, many physicians recommended against vaccination with dpt. as a result, pertussis incidence increased with substantial mortality rates. this led to a reappraisal of the immunization program, with insti-tution of incentive payments to general practitioners for completion of vaccination schedules. as a result of these measures, vaccination coverage, with resulting pertussis control, improved dramatically in the united kingdom. pertussis continues to be a public health threat and recurs wherever there is inadequate immunization in infancy. a new acellular vaccine is ready for widespread use and will be safer with fewer and less severe reactions in infants, increasing the potential for improved confidence and support for routine vaccination. use of the new vaccine is spreading in the united states and forms part of the u.s. recommended vaccination schedule. tetanus. tetanus is an acute disease caused by an exotoxin of the tetanus bacillus (clostridium tetani) which grows anaerobically at the site of an injury. the bacillus is universally present in the environment and enters the human body via penetrating injuries. following an incubation period of - days, it causes an acute condition of painful muscular contractions. unless there is modem medical care available, patients are at risk of high case fatality rates of - % (highest in infants and the elderly). antitetanus serum (ats) was discovered in and during world war i, ats contributed to saving the lives of many thousands of wounded soldiers. tetanus toxoid was developed in . the organism, because of its universal presence in the environment, cannot be eradicated. however, the disease can be controlled by effective immunization of every child during infancy and school age. adults should receive routine boosters of tetanus toxoid once very decade. newborns are infected by tetanus spores (tetanus neonatorum) where unsanitary conditions or practices are present. it can occur when traditional birth attendants at home deliveries use unclean instruments to sever the umbilical cord, or dress the severed cord with contaminated material. tetanus neonatorum remains a serious public health problem in developing countries. immunization of pregnant women and women of childbearing age is reducing the problem by conferring passive immunity to the newborn. the training of traditional birth attendants in hygienic practice and the use of medically supervised birth centers for delivery also decreases the incidence of tetanus neonastorum. elimination of tetanus neonatorum by the year was made a health target by the world summit of children in . in that year, the number of deaths from neonatal tetanus was reported by unicef as , infants worldwide, declining to , in . immunization of pregnant women increased from under % in to % in - . despite progress, coverage is still too low to achieve the target of elimination. poliomyelitis. polio virus infection may be asymptomatic or cause an acute nonspecific febrile illness. it may reach more severe forms of aseptic meningitis and acute flaccid paralysis with long-term residual paralysis or death during the acute phase. poliomyelitis is transmitted mainly by direct person-to-person contact, but also via sewage contamination. large-scale epidemics of disease, with attendant paralysis and death, occurred in industrialized countries in the s and s, engendering widespread fear and panic and thousands of clinical cases of "infantile paralysis". growth of the poliovirus by john enders and colleagues in tissue culture in led to development of the first inactivated polio vaccine by jonas salk in the mid- s and gave hope and considerable success in the control of the disease. the development of the live attenuated oral poliomyelitis vaccine by albert sabin, licensed in , added a new dimension to its control because of the effectiveness, low cost, and ease of administration of the vaccine. the two vaccines in their more modern forms, enhanced strength inactivated polio vaccine (eipv), and triple oral polio vaccine (topv), have been used in different settings with great success. oral polio vaccine (opv) induces both humoral and cellular, including intestinal, immunity. the presence of opv in the environment by contact with immunized infants and via excreta of immunized persons in the sewage gives a booster effect in the community. immunization using opv, in both routine and national immunization days (nids) has proven effective in dramatically reducing poliomyelitis and circulation of the wild virus in many parts of the world. use of the enhanced strength ipv (eipv) produces early and high levels of circulating antibodies, as well as protecting against the vaccine-associated disease. in rare cases opv can cause vaccine-associated paralytic poliomyelitis (vapp), with a risk of case per , with initial doses, and case per over million with subsequent doses. approximately eight to ten cases of vapp occur annually in the united states, with clinical, ethical, and legal implications. use of ipv as initial protection eliminates this problem. experience in gaza and the west bank in the s and s, and later in israel, showed that a combination of ipv and opv is effective in overcoming endemic and imported poliovirus. opv requires multiple doses to achieve protective antibody levels. where there are many enteroviruses in the environment, as is the case in most developing countries, interference in the uptake of opv may result in cases of paralytic poliomeylitis among persons who have received or even doses of adequate opv. controversy as to the relative advantages of each vaccine continues. the opv program of mass repeated vaccination in control of poliomyelitis in the americas established the primacy of opv in practical public health, and the momentum to eradicate poliomyelitis is building. a combined schedule of ipv and opv would eliminate the wild virus and protect against vaccine-associated disease. the sequential use of ipv and opv was adopted as part of the routine infant immunization program in the united states in , but ipv alone was adopted in . there are concerns that exclusive use of either vaccine alone will not lead to the desired goal of eradication of polyomyelitis. progress in global eradication of polio has been impressive. global coverage of infants with three doses of opv reached % in as compared to % in . the african region of who had an increase in opv coverage from % in to % in . national immunization days (nids) were conducted in countries in and in , covering million children in . mopping up operations to reinforce coverage of children in still endemic areas is proceeding along with increased emphasis on acute flaccid paralysis (afp) monitoring. confirmed polio cases reported continued at - , per year in - . with continued national and international emphasis, and support of who, rotary international, unicef, donor countries, and others, there is a real prospect of a world without polio, if not by the year , then or shortly thereafter. measles is an acute disease caused by a virus of the paramyxovirus family. it is highly infectious with a very high ratio of clinical to subclinical case ratio ( / ). measles has a characteristic clinical presentation with fever, white spots (koplik spots) on the membranes of the mouth, and a red blotchy rash appearing on the rd- th day lasting - days. mortality rates are high in young children with compromised nutritional status, especially vitamin a deficiency. the measles virus evolved from a virus disease of cattle (rinderpest) some - years ago, becoming an important disease of humans with high mortality rates in debilitated, poorly nourished children, and significant mortality and morbidity even in industrialized countries. in the prevaccine era, measles was endemic worldwide, and even in the late s it remains one of the major childhood infectious diseases. it is one of the commonest causes of death for school age children worldwide. despite earlier predictions that measles deaths would be halved to , by , who reported . million measles deaths in that year and over million in . eradication in the first decade of the next century is a feasible goal, provided that there is an adequate international effort. measles immunization increased from under % worldwide in to % in - , but % in sub-saharan africa. single-dose immunization failed to meet control or eradication requirements even in the most developed parts of the world. a live vaccine, licensed in , was later replace by a more effective and heat stable vaccine, but still with a primary vaccination failure rate (i.e., fails to produce protective antibodies) of - %, and secondary failure rate (i.e., produces antibodies but protection is lost over time) of %. a two-dose policy incorporates a booster dose, usually at school-age, in addition to maximum feasible infant coverage of children in the - month period (timing varies in different countries). catch-up campaigns among schoolage children should be carried out until the routine two-dose policy has time to take full effect. nearly universal primary education in developing countries, offers an opportunity for mass coverage of school age children with a second dose of measles and a resulting increase of herd immunity to reduce the transmission of the virus. the two-dose policy adopted in many countries, should be supplemented with catch-up campaigns in schools to provide the booster effect for those previously immunized and to cover those previously unimmunized, especially in developing countries. the cdc considers that domestic transmission in the united states has been interrupted and that most localized outbreaks were traceable to imported cases. south america and the caribbean countries are now considered free of indigenous measles, based on their successful use of nids, although a large epidemic occurred in in brazil. it now appears that eradication has become a feasible target during the early part of the next century, with a strategy of levels of coverage in in-fancy with a two-dose policy, supplemented by catch-up campaigns to older children and young adults, and outbreak control. mumps. mumps is an acute viral disease characterized by fever, swelling, and tenderness usually of the parotid glands, but also other glands. the incubation period ranges between and days. orchitis, or inflammation of the testicles, occurs in - % of postpubertal males and oophoritis, or inflammation of the ovaries, in % of postpubertal females. sterility is an extremely rare result of mumps. central nervous system involvement can occur in the form of aseptic meningitis, almost always without sequelae. encephalitis is reported in - per , cases with an overall case fatality rate of . %. pancreatitis, neuritis, nerve deafness, mastiffs, nephritis, thyroiditis, and pericarditis, although rare, may occur. most persons born before are immune to the disease, because of the nearly universal exposure to the disease before that time. the live attenuated vaccine introduced in the united states in is available as a single vaccine or in combination with measles and rubella as the measlesmumps-rubella (mmr) vaccine. it provides long-lasting immunity in % of cases. mumps vaccine is now recommended in a two-dose policy with the first dose of mmr given between and months of age and a second dose given either at school entry or in early adolescence. mmr in two doses is now standard policy in the united sates, sweden, canada, israel, the united kingdom, and other countries. the incidence of mumps has consequently declined rapidly. local eradication of this disease is worthwhile and should be part of a basic international immunization program. rubella. rubella (german measles) is generally a mild viral disease with lymphadenopathy and a diffuse, raised red rash. low grade fever, malaise, coryza, and lymphadenopathy characterize the prodromal period. the incubation period is usually - days. differentiation from scarlet fever, measles, or other febrile diseases with rash may require laboratory testing and recovery of the virus from nasopharyngeal, blood, stool, and urine specimens. in , norman gregg, an australian ophthalmologist, noted an epidemic of cases of congenital cataract in newborns associated with a history of rubella in the mother during the first trimester. subsequent investigation demonstrated that intrauterine death, spontaneous abortion, and congenital anomalies occur commonly when rubella occurs early in pregnancy. congenital rubella syndrome (crs) occurs with single or multiple congenital anomalies including deafness, cataracts, microophthalmia, congenital glaucoma, microcephaly, meningoencephalitis, congenital heart defects, and others. moderate and severe cases are recognizable at birth, but mild cases may not be detected for months or years after birth. insulin-dependent diabetes is suspected as a late sequela of congenital rubella. each case of crs is estimated to cost some $ , in health care costs during the patient's lifetime. prior to availability of the attenuated live rubella vaccine in , the disease was universally endemic, with epidemics or peak incidence every - years. in unvaccinated populations, rubella is primarily a disease of childhood. in areas where children are well vaccinated, adolescent and young adult infection is more apparent, with epidemics in institutions, colleges, and among military personnel. a sharp reduction of rubella cases was seen in the united states following introduction of the vaccine in , but increased in , following rubella epidemics in - . a further reduction in cases was followed by a sharp upswing of rubella and crs in [ ] [ ] [ ] . an outbreak of rubella among the amish in the united states, who refuse immunization on religious grounds, resulted in cases of crs in . it is now thought that vaccination of sufficient numbers in the united states reduced circulation of the virus and protected most vulnerable groups in the population. in the past, immunization policy in some countries was to vaccinate school girls aged to protect them for the period of fertility. the current approach is to give a routine dose of mmr in early childhood, followed by a second dose in early school age to reduce the pool of susceptible persons. women of reproductive age should be tested to confirm immunity before pregnancy and immunized if not already immune. should a woman become infected during pregnancy, termination of pregnancy previously recommended is now managed with hyperimmune globulin. the infection of pregnant women during their first trimester of pregnancy is the primary public health implication of rubella. the emotional and financial burden of crs, including the cost of treatment of its congenital defects, makes this vaccination program cost-effective. its inclusion in a modem immunization program is fully justified. elimination of crs syndrome should be one of the primary goals of a program for prevention of vaccine-preventable disease in developed and developing countries. adoption of mmr and the two-dose policy will gradually lead to eradication of rubella and rubella syndrome. viral hepatitis. viral hepatitis is a group of diseases of increasing public health importance due to their large scale worldwide prevalence, their serious consequences, and our increasing ability to take preventive action. viral hepatic infectious diseases each have specific etiologic, clinical, epidemiologic, serologic, and pathologic characteristics. they have important short-and long-term sequelae. vaccine development is of high priority for control and ultimate eradication. hepatitis a. hepatitis a (hav) was previously known as infectious hepatitis or epidemic jaundice. hav is mainly transmitted by the fecal-oral route. clinical severity varies from a mild illness of - weeks to a debilitating illness lasting several months. the norm is complete recovery within weeks, but a fulminating or even fatal hepatitis can occur. severity of the disease worsens with increasing age. hav is sporadic/endemic worldwide. improving sanitation raises the age of exposure, with accompanying complications. it now occurs particularly in persons from industrialized countries when exposed to situations of poor hygiene, or among young adults when traveling to areas where the disease is en-demic. common source outbreaks occur in school-aged children and young adults from case contact, or from food contaminated by infected handlers. hepatitis a may be a serious public health problem in a disaster situation. prevention involves improving personal and community hygiene, with safe chlorinated water and proper food handling. hepatitis a vaccine has been recently licensed for use in the united states, and will probably soon be recommended for routine vaccination programs, as well as for persons traveling to endemic areas. hepatitis b. hepatitis b (hbv) once called serum jaundice, was thought to be transmitted only by injections of blood or blood products. it is now known to be present in all body fluids and easily transmissible by household and sexual contact, perinatal spread from mother to newborn, and between toddlers. however, it is not spread by the oral-fecal route. hepatitis b virus is endemic worldwide and is especially prevalent in developing countries. carrier status with persistent viremia varies from < % of adults in north america to % in some parts of the world. carders have detectable levels of hbsag, the surface antigen (i.e., australian antigen), in their blood. high risk groups in developed countries include intravenous drug users, homosexual men, persons with high numbers of sexual partners, those receiving tattoos, body piercing or acupuncture treatments, and residents or staff of institutions such as group homes and prisons. immunocompromised and hemodialysis patients are commonly carders of hbv. hbv may also be spread in a health system by use of inadequately sterilized reusable syringes, as in china and the former soviet union. transmission is reduced by screening blood and blood products for hbsag and strict technique for handling blood and body fluids in health settings. hbv is clinically recognizable in less than % of infected children but is apparent in - % of infected adults. clinically hbv has an insidious onset with anorexia, abdominal discomfort, nausea, vomiting, and jaundice. the disease can vary in severity from subclinical, very mild to fulminating liver necrosis, and death. it is a major cause of primary liver cancer, chronic liver disease, and liver failure, all devastating to health and expensive to treat. hepatitis b virus is considered to be the cause of % of primary cancer of the liver in the world and the most common carcinogen after cigarette smoking. the who estimates that more than billion people alive today have been infected with hbv. it is also estimated that million persons are chronic carriers of hbv, with an estimated - . million deaths per year from cirrhosis or primary liver cancer. this makes hepatitis b control a vital issue in the revision of health priorities in many countries. strict discipline in blood banks and testing of all blood donations for hbv, as well as hiv, and hepatitis c, is mandatory, with destruction of those with positive tests. contacts should be immunized following exposure with hbv immunoglobulin and hbv vaccine. the inexpensive recombinant hbv vaccine should be adopted by all countries and included in routine vaccination of infants. catch-up immunization for older children is also desirable. immunization programs should include those exposed at work, such as health, prison, or sex workers and adults in group settings. hbv immunization has been included in who's epi-plus expanded program of immunization. hepatitis c. first identified in , and previously known as non-a, non-b hepatitis, hepatitis c (hcv) has an insidious onset with jaundice, fatigue, abdominal pain, nausea, and vomiting. it may cause mild to moderate illness, but chronicity is common going on to cirrhosis and liver failure. the cdc estimates that million americans are chronically infected with hcv, with - , resulting deaths per annum, and the main cause of liver transplants. hcv is transmitted most commonly in blood products, but also among injecting drug users ( % of intravenous drug users were hcv positive in a vancouver study in ), and is also a risk for health workers. the disease may also occur in dialysis centers and other medical situations. person-to-person spread is unclear. prevention of transmission includes routine testing of blood donations, antiviral treatment of blood products, needle exchange programs, and hygiene. the who in has declared hepatitis prevention as a major public health crisis, with an estimated million persons infected worldwide ( ) , stressing that this "silent epidemic" is being neglected and that screening of blood products is vital to reduce transmission of this disease as for hiu hcv is a major cause of chronic cirrhosis and liver cancer. no vaccine is available at present, but an experimental vaccine is undergoing field trials. interferon and ribavirin treatment is reportedly effective in % of cases. hepatitis d. hepatitis d virus (hdv) also known as delta hepatitis, may be self-limiting or progress to chronic hepatitis. it is caused by a viruslike particle which infects cells along with hbv as a coinfection or in chronic carriers of hbv. hdv occurs worldwide in the same groups at risk for hbv. it also occurs in epidemics and is endemic in south america, africa, and among drug users. prevention is by measures similar to those for hbv. management for hdv is by passive immunity with immunoglobulin for contacts and high risk groups, and should include hbv vaccination as the diseases often coincide. there is currently no vaccine for hdv. hepatitis e. hepatitis e virus has an epidemiological and clinical course similar to that of hav. there is no evidence of a chronic form of hev. one striking characteristic of hev is its high mortality rate among pregnant women. the disease is caused by a viruslike particle with an incubation period of - days and is most common in young adults. sporadic cases as well as epidemics have been identified in india, pakistan, burma, china, russia, mexico, and north africa. hev results from waterborne epidemics or as sporadic cases in areas with poor hygiene, spread via the oral-fecal route. it is a hazard in disaster situations with crowding and poor sanitary conditions. prevention is by safe management of water supplies and sanitation. disease management is supportive care; passive immunization is not helpful and no vaccine is currently available. teria which causes meningitis and other serious infections in children under months of age. before the introduction of effective vaccines, as many as in children developed invasive hib infection. two-thirds of these had hib meningitis, with a case fatality rate of - %. long-term sequelae such as hearing impairment and neurological deficits occurred in - % of survivors. the first hib vaccine was licensed in , based on capsular material from the bacteria. extensive clinical trials in finland demonstrated a high degree of efficacy, but less impressive results were in seen in postmarketing efficacy studies. by , a conjugate vaccine based on an additional protein cell capsular factor capable of enhancing the immunologic response was introduced. several conjugate vaccines are now available. the conjugate vaccines are now combined with dpt as their schedule is simultaneous with that of the dpt. although the hib vaccine has been found to be cost-effective, despite initially being as costly as all the basic vaccines combined (i.e., dpt, opv, mmr, and hbv). for this reason, its use thus far has been limited to industrialized countries. the vaccine is a valuable addition to the immunologic armamentarium. it showed dramatic results in local eradication of this serious early childhood infection in a number of european countries and a sharp reduction in the united states. impressive field trials in the gambia showed a sharp reduction in mortality from invasive streptococcal diseases. the price of the vaccine has also fallen dramatically since the mid s. as a result, in , the world health organization recommended inclusion of hib vaccine in routine immunization programs in developing countries. influenza. influenza is an acute viral respiratory illness characterized by fever, headache, myalgia, prostration, and cough. transmission is rapid by close contact with infected individuals and by airborne particles with an incubation period of - days. it is generally mild and self-limited with recovery in - days. however, in certain population groups, such as the elderly and chronically ill, infection can lead to severe sequelae. gastrointestinal symptoms commonly occur in children. during epidemics, mortality rates from respiratory diseases increase because of the large numbers of persons affected, although the case fatality rates are generally low. over the past century, influenza pandemics have occurred in , , , and , while epidemics are annual events. the influenza pandemic of caused millions of deaths among young adults, by some estimates killing more than had died in world war i. it was the fear of recurrence of this pandemic which led the cdc to launch a massive immunization program in the united states in to prevent swine flu (the virus was a strain antigenically similar to that of the pandemic influenza) from spreading from an isolated outbreak in an army camp. the effort was stopped after millions of persons were immunized with an urgently produced vaccine when serious reactions occurred (guillain-barre syndrome, (i.e., a type of paralysis), and when no further cases of swine flu were seen. this demonstrated the difficulty of extrapolating scenarios from a historical experience. each year, epidemiologic services of the who and collaborating centers such as the cdc recommend which strains should be used in vaccine preparation for use among susceptible population groups. these vaccines are prepared with the current anticipated epidemic strains. the three main types of influenza (a, b, and c) have different epidemiological characteristics. type a and its subtypes, which are subject to antigenic shift, are associated with widespread epidemics and pandemics. type b undergoes antigenic drift and is associated with less widespread epidemics. influenza type c is even more localized. active immunization against the prevailing wild strain of influenza virus produces a - % level of protection in high risk groups. the benefits of annual immunization outweigh the costs, and it has proven to be effective in reducing cases of influenza and its secondary complications such as pneumonia and death from respiratory complications in high-risk groups. pneumococcal disease. pneumococcal diseases, which are caused by streptococcus pneumoniae, include pneumonia, meningitis, and otitis media. the capsular types of pneumococci selected out of known types of the organism for the vaccine are those responsible for % of pneumococcal pneumonia cases and - % of all pneumonia cases in the united states, and are responsible for some , deaths per year. this vaccine has been found to be cost-effective for high risk groups, including persons with chronic disease, hiv carriers, patients whose spleens were removed, the elderly, and those with immunosuppressive conditions. it should be included in preventive-oriented health programs, especially for long-term care of the chronically ill. because pneumococci cause bacterial meningitis, pneumococcal vaccine may be a future candidate for use in routine immunization programs for children (over age ). varicella is an acute, generalized virus disease caused by the varicella zoster virus (vzv). despite its reputation as an innocuous disease of childhood, varicella patients can be quite ill. a mild fever and characteristic generalized red rash lasts for a few hours, followed by vesicles occurring in successive crops over various areas of the body. affected areas may include the membranes of the eyes, mouth, and respiratory tract. the disease may be so mild as to escape observation or may be quite severe, especially in adults. death can occur from viral pneumonia in adults and sepsis or encephalitis in children. neonates whose mothers develop the disease within days of delivery are at increased risk with a case fatality rate of up to %. long-term sequelae include herpes zoster or shingles with a severely painful, vesicular rash along the distribution of sensory nerves, which can last for months. its occurrence increases with age and it is primarily seen in the elderly. it can, however, occur in immunocompromised children (especially those on cancer chemotherapy), aids patients, and others. some % of a population will experience herpes zoster during their lifetimes. reye's syndrome is an increasingly rare but serious complication from varicella or influenza b. it occurs in children and affects the liver and central nervous system. congenital varicella syndrome with birth defects similar to congenital rubella syndrome has been identified recently. varicella vaccine is now recommended for routine immunization at age - months in the united states, with catch-up for children up to age years and for occupationally exposed persons in health or child care settings. varicella vaccine is also recommended for nonpregnant women of child bearing years. cost-benefit studies indicate a : ratio if both direct and indirect costs are included (see chapter ). varicella vaccine is likely to be added to a "cocktail vaccine" containing dpt, polio (ipv), and hib. meningococcal meningitis. meningococcal meningitis, caused by the bacterium neisseria meningitides, is characterized by headache, fever, neck stiffness, delirium, coma, and/or convulsions. the incubation period is - days. it has a case fatality rate of - % if treated early and adequately, but rises up to % in the absence of treatment. there are several important strains (a, b, c, x, y, and z). serogroups a and c are the main causes of epidemics, with b causing sporadic cases and local outbreaks. transmission is by direct contact and droplet spread. meningitis (group a) is common in sub-saharan african countries, but epidemics have occurred worldwide. during epidemics, children, teenagers, and young adults are the most severely affected. in developed countries, outbreaks occur most frequently in military and student populations. in , meningococcal meningitis spread widely in the "meningitis belt" in central africa. epidemic control is achieved by mass chemoprophylaxis with antibiotics (e.g., rifampin or sulfa drugs) among case contacts, although the emergence of resistant strains is a concern. vaccines against serotypes a and c (bivalent) or a, c, w, and y are available. their use is effective in epidemic control and prevention institutions and military recruits, especially for a and c serogroups. vaccination is one of the key modalities of primary prevention. immunization is cost-effective and prevents wide-scale disease and death, with high levels of safety. despite the general consensus in public health regarding the central role of vaccination, there are many areas of controversy and unfulfilled expectations. a vaccination program should aim at % coverage at appropriate times, including infants, school children, and adults. immunization policy should be adapted from current international standards applying the best available program to national circumstances and financial capacities (table . ). public health personnel with expertise in vaccine-preventable disease control are needed to advise ministries of health and the practicing pediatric community on current issues in vaccination and to monitor implementation and evolution of control programs. controversies and changing views are common to immunization policy, so that discussions must be conducted on a continuing basis. policy should be under continuing review by a ministerially appointed national immunization advisory committee, including professionals from public health, academia, immunology, laboratory sciences, economics, and relevant clinical fields. bduring , the recommendation for polio virus was changed to doses of ipv in infancy. vaccine supply should be adequate and continuous. supplies should be ordered from known manufacturers meeting international standards of good manufacturing practice. all batches should be tested for safety and efficacy prior to release for use. there should be an adequate and continuously monitored cold chain to protect against high temperatures for heat labile vaccines, sera, and other active biological preparations. the cold chain should include all stages of storage, transport, and maintenance at the site of usage. only disposable syringes should be used in vaccination programs to prevent any possible transmission of blood-borne infection. a vaccination program depends on a readily available service with no barriers or unnecessary prerequisites, free to parents or with a minimum fee, to administer vaccines in disposable syringes by properly trained individuals using patientoriented and community-oriented approaches. ongoing education and training on current immunization practices are needed. incentive payments by insuring agency or managed care systems promote complete, on-time coverage. all clinical encounters should be used to screen, immunize, and educate parents/guardians. contraindications to vaccination are very few; vaccines may be given even during mild illness with or without fever, during antibiotic therapy, during convalescence from illness, following recent exposure to an infectious disease, and to persons having a history of mild/moderate local reactions, convulsions, or family history of sudden infant death syndrome (sids). simultaneous administration of vaccines and vaccine "cocktails" reduces the number of visits and thereby improves coverage; there are no known interferences between vaccine antigens. accurate and complete recording with computerization of records with automatic reminders helps promote compliance, as does co-scheduling of immunization appointments with other services. adverse events should be reported promptly, accurately, and completely. a tracking system should operate with reminders of upcoming or overdue immunizations; use mail, telephone, and home visits, especially for high risk families, with semiannual audits to assess coverage and review patient records in the population served to determine the percentage of children covered by second birthday. tracking should identify children needing completion of the immunization schedule and assess the quality of documentation. it is important to maintain up-to-date, easily retrievable medical protocols where vaccines are administered, noting vaccine dosage, contraindications, and management of adverse events. all health care providers and managers should be trained in education, promotion, and management of immunization policy. health education should target parents as well as the general public. monitoring of vaccines used and children immunized, individually and by category of vaccination can be facilitated by computerization of immunization records, or regular manual review of child care records. where immunization is done by physicians in private practice, as in the united states, determination of coverage is by periodic surveys. inspection of vaccines for safety, purity, potency, and standards is part of the regulatory function. vaccines are defined as biological products and are therefore subject to regulation by national health authorities. in the united states, this comes under the legislative authority of the public health service act, as well as the food, drug and cosmetics act, with applicable regulations in the code of federal regulations. the federal agency empowered to carry out this regulatory function is the center for drugs and biologics of the federal food and drug administration. litigation regarding adverse effects of vaccines led to inflation of legal costs and efforts to limit court settlements. the u.s. federal government enacted the child vaccine injury act of . this legislation requires providers to document vaccines given and to report on complications or reactions. it was intended to pay benefits to persons injured by vaccines faster and by means of a less expensive procedure than a civil suit for resolving claims. using this no-fault system, petitioners do not need to prove that manufacturers or vaccine givers were at fault. they must only prove that the vaccine is related to the injury in order to receive compensation. the vaccines covered by this legislation include dtp, mmr, opv, and ipv. development of vaccines from jenner in eighteenth century to the advent of recombinant hepatitis b vaccine in , and of vaccines for acellular pertussis, varicella, hepatitis a, and rotavirus in the s, has provided one of the pillars of public health and led to enormous savings of human life. vaccines for viral in-fections in humans for hiv, respiratory syncytial virus, papilloma, epstein-barr virus, dengue fever, and hantavirus are under intense research with genetic approaches using recombinant techniques. the potential for the future of vaccines will be greatly influenced by scientific advances in genetic engineering, with potential for development of vaccines attached to bacteria or protein in plants, which may be given in combination for an increasing range of organisms or their harmful products. recombinant dna technology has revolutionized basic and biomedical research since the s. the industry of biotechnology has produced important diagnostic tests, such as for hiv, with great potential for vaccine development. traditional whole organism vaccines, alive or killed, may contain toxic products that may cause mild to severe reactions. subunit vaccines are prepared from components of a whole organism. this avoids the use of live organisms that can cause the disease or create toxic products which cause reactions. subunit vaccines traditionally prepared by inactivation of partially purified toxins are costly, difficult to prepare, and weakly immunogenic. recombinant techniques are an important development for production of new whole cell or subunit vaccines that are safe, inexpensive, and more productive of antibodies than other approaches. their potential contribution to the future of immunology is enormous. molecular biology and genetic engineering have made it feasible to create new, improved, and less costly vaccines. new vaccines should be inexpensive, easily administered, capable of being stored and transported without refrigeration, and given orally. the search for inexpensive and effective vaccines for groups of viruses causing diarrheal diseases led to development of the rotavirus vaccine. some "edible" research focuses on the genetic programming of plants to produce vaccines and dna. vaccine manufacturers, who spend huge sums of money and years of research on new products, tend to work on those which will bring great financial rewards for the company and are critical to the local health care community. this has led to less effort being made in developing vaccines for diseases such as malaria. yet industry plays a crucial role for continued progress in the field. since the eradication of smallpox, much attention has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and some tropical diseases, such as malaria and dracunculiasis. eradication is defined as the achievement of a situation whereby no further cases of a disease occur anywhere and continued control measures are unnecessary. reducing epidemics of infectious diseases, through control and eradication in selected areas or target groups, can in certain instances achieve eradication of the disease. local eradication can be achieved where domestic circulation of an organism is interrupted with cases occurring from importation only. this requires a strong, sustained immunization program with adaptation to meet needs of importation of carriers and changing epidemiologic patterns. smallpox was one of the major pandemic diseases of the middle ages and its recorded history goes back to antiquity. prevention of smallpox was discussed in ancient china by ho kung (circe ao ), and inoculation against the disease was practiced there from the eleventh century ad. prevention was carried out by nasal inhalation of powdered dried smallpox scabs. exposure of children to smallpox when the mortality rate was lowest assumed a weakened form of the disease, and it was observed that a person could only have smallpox once in a lifetime. isolation and quarantine were widely practiced in europe during the sixteenth and seventeenth centuries. variolation was the practice of inoculating youngsters with material from scabs of pustules from mild cases of smallpox in the hope that they would develop a mild form of the disease. although this practice was associated with substantial mortality, it was widely adopted because mortality from variolation was well below that of smallpox acquired during epidemics. introduced into england in (see chapter ) it was commonly practiced as a lucrative medical specialty during the eighteenth century. in the s, variolation was also introduced into the american colonies, russia, and subsequently into sweden and denmark. despite all efforts, in the early eighteenth century smallpox was a leading cause of death in all age groups. toward the end of the eighteenth century an estimated , persons died annually from smallpox in europe. vaccination, or the use of cowpox vaccinia virus to protect against smallpox, was initiated late in the eighteenth century. in , a cattle breeder in yorkshire, england, inoculated his wife and two children with cowpox to protect them during a smallpox epidemic. in , edward jenner, an english country general practitioner, experimented with inoculation from a milkmaid's cowpox pustule to a healthy youngster, who subsequently proved resistant to smallpox by variolation (see chapter ). vaccination, the deliberate inoculation of cowpox material, was slow to be adopted universally, but by , over , persons in england were vaccinated. vaccination gathered support in the nineteenth century in military establishments, and in some countries which adopted it universally. opposition to vaccination remained strong for nearly a century based on religious grounds, observed failures of vaccination to give lifelong immunity, and because it was seen as an infringement of the state on the rights of the individual. often the protest was led by medical variolationists whose medical practice and large incomes were threatened by the mass movement to vaccination. resistance was also offered by "sanitarians" who opposed the germ theory and thought cleanliness was the best method of prevention. universal vaccination was increasingly adopted in europe and america in the early nineteenth century and eradication of smallpox in developed countries was achieved by the mid twentieth century. in , the soviet union proposed to the world health assembly a program to eradicate smallpox internationally and subsequently donated million doses of vaccine per year as part of the million needed to promote vaccination of at least % of the world population. in , who adopted a target for the eradication of smallpox. a program was developed which included a massive increase in coverage to reduce the circulation of the virus through person-to-person contact. where smallpox was endemic, with a substantial number of unvaccinated persons, the aim of the mass vaccination phase was % coverage. increasing vaccination coverage in developing countries reduced the disease to periodic and increasingly localized outbreaks. in , countries were considered endemic for smallpox, and another experienced importation of cases. by , the number of endemic countries was down to , and by only countries were still endemic, including india, pakistan, bangladesh, and nepal. in these countries, a new strategy was needed, based on a search for cases and vaccination of all contacts, working with a case incidence below per , . the program then moved into the consolidation phase, with emphasis on vaccination of newborns and new arrivals. surveillance and case detection were improved with case contact or risk group vaccination. the maintenance phase began when surveillance and reporting were switched to the national or regional health service with intensive follow-up of any suspect case. the mass epidemic era had been controlled by mass vaccination, reducing the total burden of the disease, but eradication required the isolation of individual cases with vaccination of potential contacts. technical innovations greatly eased the problems associated with mass vaccination worldwide. during the s, there was wide variation in sources of smallpox vaccine. in the s, efforts to standardize and further attenuate the strains used reduced complication rates from vaccinations. the development of lyophilization (freeze-drying) of the vaccine in england in the s made a heat-stable vaccine that could be effective in tropical field conditions in developing countries. the invention of the bifurcated needle (bernard rubin ) allowed for easier and more widespread vaccination by lesser trained personnel in remote areas. the net result of these innovations was increased world coverage and a reduction in the spread of the disease. smallpox became more and more confined by increasing herd immunity, thus allowing transition to the phase of monitoring and isolation of individual cases. in the last case of smallpox was identified in somalia, and in the who declared the disease eradicated. no subsequent cases have been found except for several associated with a laboratory accident in the united kingdom in . the who recommends that the last stores of smallpox virus should be destroyed in . the cost of the eradication program was $ million or $ million per year. worldwide savings are estimated at $ billion annually. this monumental public health achievement set the precedent for eradication of other infectious diseases. the world health assembly decided to destroy the last two remaining stocks of the smallpox virus in atlanta and moscow in . destruction of the remaining stock was delayed in to because of concern that illegal stocks may be held by some states or potential bioterrorists for potential use in weapons of mass destruction, concern regarding the appearance of monkeypox and a wish to use the virus for further research. in , the who established a target of eradication of poliomyelitis by the year . global immunization coverage with three doses of opv increased from some % in to over % in , with a slight decline in the period - . support from member countries and international agencies such as unicef and rotary international has led to widescale increases in immunization coverage throughout many parts of the world. the world health organization promotes use of opv only as part of routine infant immunization or national immunization days (nids). this strategy has been successful in the americas and in china, but india and the middle east remain problematic. eradication of wild poliomyelitis by the year will require flexibility in vaccination strategies and may require the combined approach, using opv and ipv, as adopted in the united states in to prevent vaccine-associated clinical cases. the combination of opv and ipv may be needed where enteric disease is common and leads to interference in opv uptake, especially in tropical areas where endemic poliovirus and diarrheal diseases are still found. the world bank estimated that achievement of global eradication would save $ million annually in the united states alone. since the eradication of smallpox, discussion has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and tropical diseases such as malaria and dracunculiasis. eradication of malaria was thought to be possible in the s when major gains were seen in malaria control by aggressive case environmental control, case finding, and management. however, lack of sustained vector control and an effective vaccine has prevented global eradication. malaria control suffered serious setbacks because of failure in political resolve and capacity to continue support needed for necessary programs. in the s and s, control efforts were not sustained in many countries, and a dreadful comeback of the disease occurred in africa and asia in the s. the emergence of mosquitoes resistant to insecticides, and malarial strains resistant to antimalarial drugs, have made malaria control even more difficult and expensive. renewed effort in malaria control may require new approaches. use of community health workers (chws) in small villages in highly endemic regions of colombia resulted in a major drop in malaria mortality during the s. the chws investigate suspect cases by taking clinical histories and blood smears. . scientific feasibility a. epidemiologic vulnerability; lack of nonhuman reservoir, ease of spread, no natural immunity, relapse potential; b. effective practical intervention available; vaccine or other primary preventive or curative treatment, or vectoricide that is safe, inexpensive, long lasting, and easily used in the field; c. demonstrated feasibility of elimination in specific locations, such as an island or other geographic unit. . political will/popular support a. they examine smears for malaria parasites and a diagnosis is made. therapy is instituted and the patient is followed. quality control monitoring shows high levels of accuracy in reading of slides compared to professional laboratories. in the late s, there was widespread discussion in the literature of the potential for eradication of measles and tb. measles eradication was set back as breakthrough epidemics occurred in the united states, canada, and many other countries during the s and early s, but regional eradication was achieved combining the two-dose policy with catch-up campaigns for older children or in national immunization days, as in the caribbean countries. tuberculosis has also increased in the united states and several european countries for the first time in many decades. unrealistic expectations can lead to inappropriate assessments and policy when confounding factors alter the epidemiologic course of events. such is the case with tb, where control and eradication have receded from the picture. this deadly disease has returned to developed countries, partly in association with the hiv infection and multiple-drug-resistant strains, as well as homelessness, rising prison populations, poverty, and other deleterious social conditions. directly observed therapy is an important recent breakthrough, more effective in use of available technology and will play a major role in tb control in the twenty-first century. a decade after the eradication of smallpox was achieved, the international task force for disease eradication (itfde) was established to systematically evaluate the potential for global eradicability of candidate diseases. its goals were to identify specific barriers to the eradication of these diseases that might be surmountable and to promote eradication efforts. the subject of eradication versus control of infectious diseases if of central public health importance as technology expands the armamentarium of immunization and vector control into the twenty-first century. the control of epidemics, followed by interruption of transmission and ultimately eradication, will save countless lives and prevent serious damage to children throughout the world. the smallpox achievement, momentous in itself, points to the potential for the eradication of other deadly diseases. the skillful use of existing and new technology is an important priority in the new public health. flexibility and adaptability are as vital as resources and personnel. selecting diseases for eradication is not purely a professional issue of resources such as vaccines and manpower, organization and financing. it is also a matter of political will and perception of the burden of disease. there will be many controversies. the selection of polio for eradication while deferring measles when polio kills few and measles kills many may be questioned. the cdc published criteria for selection of disease for eradication are shown in box . . the who, in a review of health targets in the field of infectious disease control for the twenty-first century, selected the following targets: eradication of chagas' disease by ; eradication of neonatal tetanus by ; eradication of leprosy by ; eradication of measles by ; eradication of trachoma by ; reversing the current trend of increasing tuberculosis and hiv/aids. in , a conference in atlanta, georgia, reviewed the subject, which is still very much in a state of flux. table . summarizes the selection of diseases which are presently seen as controllable and those considered to be potentially eradicable. the subject will be under review in the years ahead. mycobacterium tuberculosis in humans and m. bovis in cattle. the disease is primarily found in humans, but it is also a disease of cattle and occasionally other primates in certain regions of the world. it is transmitted via airborne droplet nuclei from persons with pulmonary or laryngeal tb during coughing, sneezing, talking, or singing. the initial infection may go unnoticed, but tuberculin sensitivity appears within a few weeks. about % of those infected enter a latent phase with a lifelong risk of reactivation. approximately % go from initial infection to pulmonary tb. less commonly, the infection develops as extrapulmonary tb, involving meninges, lymph nodes, pleura, pericardium, bones, kidneys, or other organs. untreated, about half of the patients with active tb will die of the disease within years, but modern chemotherapy almost always results in a cure. pulmonary tb symptoms include cough and weight loss, with clinical findings on chest examination and confirmation by findings of tubercle bacilli in stained smears of sputum and, if possible, growth of the organism on culture media, and changes in the chest x-ray. tuberculosis affects people in their adult working years, with - % of cases in persons between the ages of and . its devastating effects on the work force and economic development contribute to a high cost-effectiveness for tb control. the tubercle bacillus infects approximately . billion people in the world today, causing over million cases and nearly million deaths in . during , new cases of tb included . million ( %) in southeast asia and the western pacific regions of who, with . million cases in india, and . million in indonesia. by , the incidence of tb may increase to . million new cases per year, a % increase over . between and , who estimates there were million new cases of tb, of which million cases were in association with hiv infection. during the s, an estimated million persons died of tb, including . million with hiv infection. a new and dangerous period for tb resurgence has resulted from parallel epidemiologic events: first, the advent of hiv infection and second, the occurrence of multiple drug resistant tb (mdrtb), that is, organisms resistant at least to both isoniazid (inh) and rifampicin, two mainstays of tb treatment. mdrtb can have a case fatality rate as high as %. hiv reduces cellular immunity so that people with latent tb have a high risk of activation of the disease. it is estimated that hiv negative persons have a - % lifetime risk of tb; hiv positive people have a risk of % per year of developing clinical tuberculosis. drug resistance, the long period of treatment, and the socioeconomic profile of most tb patients combine to require a new approach to therapy. directly observed treatment, short-course (dots), has shown itself to be highly effective with patients in poor self-care settings, such as the homeless, drug users, and those with aids. the strategy of dots uses community health workers to visit the patient and observes him or her taking the various medications, providing both incentive, support, and moral coercion to complete the needed to month therapy. dots has been shown to cure up to % of cases, at a cost of as little as $ per patient. it is one of the few hopes of containing the tb pandemic. in , who released a new strategy for control of tuberculosis over the next decade. the plan calls for new guidelines for control, new aid funds for developing countries, and enlistment of ngos to assist in the fight. the new guidelines stress short-term chemotherapy in well-managed programs of dots, stressing strict compliance with therapy for infectious cases with a goal of an % cure rate. even under adverse conditions, dots produces excellent results. it is one of the most cost-effective health interventions combining public health and clinical medical approaches. tuberculosis incidence in the united states decreased steadily until , increased in , and has declined again since. from to , there was an excess of , cases over the expected rate if the previous decline in case incidence had continued. this rise was largely due to the hiv/aids epidemic and the emergence of mdrtb, but also greater incidence among immigrants from areas of higher tb incidence, drug abusers, the homeless, and those with limited access to health care. this is particularly true in new york city, where mdrtb has appeared in outbreaks among prison inmates and hospital staff. from to , tb incidence in the united states declined by % and in some states, including new york, by % or more. this turnaround was due to stronger tb control programs that promptly identified persons with tb and initiated and ensured completion of appropriate therapy. aggressive staff training, outreach, and case management approaches were vital to this success. concern over rising rates among recent immigrants and the continued challenge of hiv/aids and coincidental transmission of hepatitis a, b, and c among drug users and marginal population groups show that continued support for tb control is needed. bacillus calmette-gurrin (bcg) is an attenuated strain of the tubercle bacillus used widely as a vaccination to prevent tb, especially in high incidence areas. it induces tuberculin sensitivity or an antigen-antibody reaction in which antibodies produced may be somewhat protective against the tubercle bacillus in % of vaccinees. although the support for its general use is contradictory, there is evidence from case-control and contact studies of positive protection against tb meningitis and disseminated tb in children under the age of . in some developed, low-incidence countries, it is not used routinely but selectively. it may also be used in asymptomatic hiv-positive persons or other high risk groups. the bcg vaccine for tuberculosis remains controversial. while used widely internationally, in the united states and other industrialized countries, it is thought to hinder rather than help in the fight against tb. this concern is based on the usefulness of tuberculin testing for diagnosis of the disease. where bcg has been administered, the diagnostic value of tuberculin testing is reduced, especially in the period soon after the bcg is used. studies showing equivocal benefit of bcg in preventing tuberculosis have added to the controversy. while those in the field in the united states continue to oppose the use of bcg, internationally it is still felt to be of benefit in preventing tb, primarily in children. a metaanalysis of the literature of bcg carried out by the technology assessment group at harvard school of public health concluded: on average, bcg vaccine significantly reduces the risk of tb by %. protection is observed across many populations, study designs, and forms of tb. age at vaccination did not enhance predictiveness of bcg efficacy. protection against tuberculous death, meningitis, and disseminated disease is higher than for total tb cases, although this result may reflect reduced error in disease classification rather than greater bcg efficacy. [colditz et al., jama, .] box . control of tuberculosis . identifying persons with clinically active tb; . diagnostic methods--clinical suspicion, sputum smear for bacteriologic examination, tuberculin skin testing, chest radiograph; . case finding and investigation programs in high risk groups; . contact investigation; . isolation techniques during initial therapy; . treatment, mainly ambulatory, of persons with clinically active tb; . treatment of contacts; . directly observed treatment, short-course (dots), where compliance suspect; . environmental control in treatment settings to reduce droplet infection; . educate health care providers on suspicion of tb and investigation of suspects. currently, the who recommends use of bcg as close to birth as possible as part of the expanded programme of immunization (epi). tuberculosis control remains feasible with current medical and public health methods. deterioration in its control should not lead to despair and passivity. the recent trend to successful control by dots despite the growing problem of mdrtb suggest that control and gradual reduction can be achieved by an activist, community outreach approach. the who in made tb control one of its major priorities, expressing grave concern that the mdr organism, now widely spread in countries of asia, eastern europe, and the former soviet union, may spread the disease much more widely. the disease constitutes one of the great challenges to public health at the start of the new century. acute infectious diseases caused by group a streptococci include streptococcal sore throat, scarlet fever, puerperal fever, septicemia, ersypelas, cellulitis, mastoiditis, otitis media, pneumonia, peritonsillitis (quinsy), wound infections, toxic shock syndrome, and fasciitis, the "flesh eating bacteria." streptococcus pyogenes group a include some serologically distinct types which vary in geographic location and clinical significance. transmission is by droplet, person-to-person direct contact, or by food infected by carriers. important complications from a public health point of view include acute rheumatic fever and acute glomerulonephritis, but also skin infections and pneumonia. acute rheumatic fever is a complication of strep a infection that has virtually disappeared from industrialized countries as a result of improved standards of living and antibiotic therapy. however, outbreaks were recorded in the united states in , and an increasing number of cases have been seen since . in developing countries, rheumatic fever remains a serious public health problem affecting school age children, particularly those in crowded living arrangements. longterm sequelae include disease of the mitral and aortic heart valves, which require cardiac care and surgery for repair or replacement with artificial valves. acute glomerulonephritis is a reaction to toxins of the streptococcal infection in the kidney tissue. this can result in long-term kidney failure and the need for dialysis or kidney transplantation. this disease has become far less common in the industrialized countries, but remains a public health problem in developing countries. the streptococcal diseases are controllable by early diagnosis and treatment with antibiotics. this is a major function of primary care systems. recent increases in rheumatic fever may herald a return of the problem, perhaps due to inadequate access to primary care in the united states for large sectors of the population, along with increased social hygiene problems. where access to primary care services is limited, infections with streptococci can result in a heavy burden of chronic heart and kidney disease with substantial health, emotional, and financial tolls. measures to improve access to care and pub-lic information are needed to assure rapid and effective care to prevent chronic and costly conditions. zoonoses are infectious diseases transmissible from vetebrate animals to humans. common examples of zoonoses of public health importance in nonindustrialized countries include brucellosis and rabies. in industrialized countries, salmonellosis, "mad cow disease" and influenza have reinforced the importance of relationships of animal and human health. strong cooperation between public health and veterinary public health authorities are required to monitor and to prevent such diseases. brucellosis is a disease occurring in cattle (brucella abortus), in dogs (br. cahis), in goats and sheep (br. melitensis), and in pigs (br. suis). humans are affected mainly through ingestion of contaminated milk products, by contact, or inhalation. brucellosis (also known as relapsing, undulant, malta, or mediterranean fever) is a systemic bacterial disease of acute or insidious onset characterized by fever, headache, weakness, sweating, chills, arthralgia, depression, weight loss, and generalized malaise. spread is by contact with tissues, blood, urine, vaginal discharges, but mainly by ingestion of raw milk and dairy products from infected animals. the disease may last from a few days to a year or more. complications include osteoarthritis and relapses. case fatality is under %, but disability is common and can be pronounced. the disease is primarily seen in mediterranean countries, the middle east, india, central asia, and in central and south america. brucellosis occurs primarily as an occupational disease of persons working with and in contact with tissues, blood, and urine of infected animals, especially goats and sheep. it is an occupational hazard for veterinarians, packinghouse workers, butchers, tanners, and laboratory workers. it is also transmitted to consumers of unpasteurized milk from infected animals. animal vectors include wild animals, so that eradication is virtually impossible. diagnosis is confirmed by laboratory findings of the organism in blood or other tissue samples, or with rising antibody titers in the blood, with confirmation by blood cultures. clinical cases are treated with antibiotics. epidemiologic investigation may help track down contaminated animal flocks. routine immunization of animals, monitoring of animals in high risk areas, quarantining sick animals, destroying infected animals, and pasteurizing milk and milk products prevents spread of the disease. control measures include educating farmers and the public not to use unpasteurized milk. individuals who work with animals (cattle, swine, goats, sheep, dogs, coyotes) should take special precautions when handling animal carcasses and materials. testing animals, destroying carriers, and enforcing mandatory pasteurization will restrict the spread of the disease. this is an economic as well as public health problem, requiring full cooperation between ministries of health and of agriculture. rabies is primarily a disease of animals, with a variety of wild animals serving as a reservoir for this disease, including foxes, wolves, bats, skunks, and raccoons, who may infect domestic animals such as dogs, cats, and farm animals. animal bites break the skin or mucous membrane, allowing entry of the virus from the infected saliva into the bloodstream. the incubation period of the virus is - weeks; it can be as long as several years or as short as days, so that postexposure preventive treatment is a public health emergency. the clinical disease often begins with a feeling of apprehension, headache, pyrexia, followed by muscle spasms, acute encephalitis, and death. fear of water ("hydrophobia") or fear of swallowing is a characteristic of the disease. rabies is almost always fatal within a week of onset of symptoms. the disease is estimated to cause , deaths annually, primarily in developing countries. it is uncommon in developed countries. rabies control focuses on prevention in humans, domestic animals, and wildlife. prevention in humans is based on preexposure prophylaxis for groups at risk (e.g., veterinarians, zoo workers) and postexposure immunization for persons bitten by potentially rabid animals. because reducing exposure of pets to wild animals is difficult, immunization of domestic animals is one of the most important preventive measures. prevention in domestic animals is by mandatory immunization of household pets. all domestic animals should be immunized at age months and revaccinated according to veterinary instructions. prevention in wild animals to reduce the reservoir is successful in achieving local eradication in settings where reentry from neighboring settings is limited. since , the use of oral rabies immunization has been successful in reducing the population of wild animals infected by the rabies virus. rabies eradication efforts, using aerial distribution of baits containing fox rabies vaccine in affected areas of belgium, france, germany, italy, and luxembourg, have been underway since . the number of rabies cases in these affected areas has declined by some %. switzerland is now virtually rabies-free because of this vaccination program. the potential exists for focal eradication, especially on islands or in partially restricted areas with limited possibilities of wild animal entry. livestock need not be routinely immunized against rabies, except in high risk areas. where bats are major reservoirs of the disease, as in the united states, eradication is not presently feasible. salmonella, discussed later in this chapter under diarrheal diseases, is one of the commonest of all infectious diseases among animals and is easily spread to humans via poultry, meat, eggs, and dairy products. specific antigenic types are associated with food-borne transmission to humans, causing generalized illness and gastroenteritis. severity of the disease varies widely, but the diseases can be devastating among vulnerable population groups, such as young children, the elderly, and the immunocompromised. epidemiologic investigation of common food source outbreaks may uncover hazardous food handling practices. laboratory confirmation or serotypes helps in monitoring the disease. prevention is by maintaining high standards of food hygiene in processing, inspection and regulation, food handling practices, and hygiene education. bacillus anthracis causes a bacterial infection in herbivore animals. its spores contaminate soil, worldwide. it affects humans exposed in occupational settings. transmission is cutaneous by contact, gastrointestinal by ingestion, or respiratory by inhalation. it has gained recent attention (iraq, ) as a highly potent agent for germ warfare or terrorism. limited supplies of vaccine are available. creutzfeld-jakob disease is a degenerative disease of the central nervous system linked to consumption of beef from cattle infected with bovine spongiform encephalopathy. it is transmitted by prions in animal feed prepared from contaminated animal material and in transplanted organs. this disease was identified in the united kingdom linked to infected cattle leading to a ban on british beef in many parts of the world and slaughter of large numbers of potentially contaminated animals. the tapeworm causing diphyllobothriasis (diphyllobothrium latum) is widespread in north american freshwater fish, passing from crustacean to fish to humans by eating raw freshwater fish. it is especially common among inuit peoples and may be asymptomatic or cause severe general and abdominal disorder. food hygiene (freezing and cooking of meat) is recommended; treatment is by anthelminthics. leptospiroses are a group of zoonotic bacterial diseases found worldwide in rats, raccoons, and domestic animals. it affects farmers, sewer workers, dairy and abattoir workers, veterinarians, military personnel, and miners with transmission by exposure to or ingestion of urine-contaminated water or tissues of infected animals. it is often asymptomatic or mild, but may cause generalized illness like influenza, meningitis, or encephalitis. prevention requires education of the public in self protection and immunization of workers in hazardous occupations, along with immunization and segregation of domestic animals and control of wild animals. vector-borne diseases are a group of diseases in which the infectious agent is transmitted to humans by crawling or flying insects. the vector is the intermediary between the reservoir and the host. both the vector and the host may be affected by climatic condition; mosquitoes thrive in warm, wet weather and are suppressed by cold weather; humans may wear less protective clothing in warm weather. the only important reservoir of malaria is humans. its mode of transmission is from person to person via the bite of an infected female anopheles mosquito (ronald ross, nobel prize, ) . the causative organism is a single cell parasite with four species: plasmodium vivax, p malariae, p falciparum, and p ovale. clinical symptoms are produced by the parasite invading and destroying red blood cells. the incubation period of approximately - days, depending on the specific plasmodium involved. some strains of p vivax may have a protracted incubation period of - months and even longer for p ovale. the disease can also be transmitted through infected blood transfusions. confirmation of diagnosis is by demonstrating malaria parasites on blood smears. falciparum malaria, the most serious form, presents with fever, chills, sweats, and headache. it may progress to jaundice, bleeding disorders, shock, renal or liver failure, encephalopathy, coma, and death. prompt treatment is essential. case fatality rates in untreated children and adults are above %. an untreated attack may last months. other forms of malaria may present as a nonspecific fever. relapse of the p ovale may occur up to years after initial infection; malaria may persist in chronic form for up to years. malaria control advanced during the s- s through improved chlovaquine treatment and use of ddt for vector control with optimism for eradication of the disease. however, control regressed in many developing countries as allocations for environmental control and case findings/treatment were reduced. there has also been an increase in drug resistance, so that this disease is now an extremely serious public health problem in many parts of the world. the need for a vaccine for malaria control is now more apparent than ever. the world health organization estimated that, in , sub-saharan africa (ssa) had million new malaria cases, with % of children up to age . over million deaths occur annually from malaria more than two-thirds of them in ssa. large areas, particularly in forest or savannah regions with high rainfall, are holoendemic. in higher altitudes, endemicity is lower, but epidemics do occur. chloroquine-resistant p. falciparum has spread throughout africa, accompanied by an increasing incidence of severe clinical forms of the disease. the world bank estimates that % of all disability-adjusted life years (dalys) lost per year in ssa are from malaria, which places a heavy economic burden on the health systems. in the americas, the number of cases detected has risen every year since , and the who estimates there to have been . - . million cases in . the nine most endemic countries in the americas achieved a % reduction in malaria mortality between and . southeast asian region reports some . million cases of malaria in and deaths from tb. this accounts for more than one-third of all non-african malaria cases. there is an increase in resistant strains to the major available drugs and of the mosquitoes to insecticides in use. vector control, case finding, and treatment remain the mainstay of control. use of insecticide-impregnated bed nets and curtains, and residual house spraying, and strengthened vector control activities are important, as are early diagnosis and carefully monitored treatment with monitoring for resistance. control of malaria will ultimately depend on a safe, effective, and inexpensive vaccine. attempts to develop a malaria vaccine have been unsuccessful to date due to the large number of genetic types of p. falciparum even in localized areas. a colombian-developed vaccine is being field-tested with partial effectiveness. research in vaccines for malaria has also been hampered by the fact that it is a relatively low priority for vaccine manufacturers because of the minimal potential for financial benefit. research on malaria concentrates on the pharmacological aspects of the disease because of increasing drug resistance. in , who has initiated a new campaign to "roll back malaria" and maintain the dream of eradication in the future. effective low technology interventions include community-based case finding, early treatment of good quality, insecticide use, and vector control. the use of community health workers in endemic areas, has shown promising results. local control and even eradication can be achieved with currently available technology. this requires an integration of public health and clinical approaches with strong political commitment. the rickettsia are obligate parasites, i.e., they can only replicate in living cells, but otherwise they have characteristics of bacteria. this is a group of clinically similar diseases, usually characterized by severe headache, fever, myalgia, rash, and capillary bleeding causing damage to brain, lungs, kidneys, and heart. identification is by serological testing for antibodies, but the organisms can also be cultured in laboratory animals, embryonic eggs, or in cell cultures. the organisms are transmitted by arthropod vectors such as lice, fleas, ticks, and mites. the diseases caused millions of deaths during war and famine periods prior to the advent of antibiotics. these diseases appear in nature in ways that make them impossible to eradicate, but clinical diagnosis, host protection, and vector control can help reduce the burden of disease and deal with outbreaks that may occur. public education regarding self-protection, appropriate clothing, tick removal, and localized control measures such as spraying and habitat modification are useful. epidemic typhus, first identified in , is due to rickettsia prowazekii. spread primarily by the body louse, typhus was the cause of an estimated million deaths, i.e., during war and famine, in poland and the soviet union from - . untreated, the fatality rate is - %. typhus responds well to antibiotics. it is currently largely confined to endemic foci in central africa, central asia, eastern europe, and south america. it is preventable by hygiene and pediculicides such as ddt and lindane. a vaccine is available for exposed laboratory personnel. murine typhus is a mild form of typhus due to rickettsia typhi, which is found worldwide and spread in rodent reservoirs. scrub typhus, also known as tsutsugamushi or japanese river fever, is located throughout the far east and the pacific islands, and was a serious health problem for u.s. armed forces in the pacific during world war ii. it is spread by the rickettsia tsutsugamushi and has a wide variation in case fatality according to region, organism, and age of patient. rocky mountain spotted fever is a well-known and severe form of tick-borne typhus due to rickettsia rickettsii, occurring in western north america, europe, and asia. q. fever is a tick-borne disease caused by coxiella burnetii and is worldwide in distribution, usually associated with farm workers, in both acute and chronic forms. regular anti-tick spraying of sheep, cows, and goats helps protect exposed workers. protective clothing and regular removal of body ticks help protect exposed persons. arthropod-borne viral diseases are caused by a diverse group of viruses which are transmitted between vertebrate animals (often farm animals or small rodents) and people by the bite of blood-feeding vectors such as mosquitoes, ticks, and sandflies and by direct contact with infected animal carcasses. usually the viruses have the capacity to multiply in the salivary glands of the vector, but some are carried mechanically in their mouthparts. these viruses cause acute central nervous system infections (meningoencephalitis), myocarditis, or undifferentiated viral illnesses with polyarthritis and rashes, or severe hemorrhagic febrile illnesses. arbovirus diseases are often asymptomatic in vertebrates but may be severe in humans. over antigenetically distinct arboviruses are associated with disease in humans, varying from benign fevers of short duration to severe hemmorhagic fevers. each has a specific geographic location, vector, clinical, and virologic characteristics. they are of international public health importance because of the potential for spread via natural phenomena and modem rapid transportation of vectors and persons incubating the disease or ill with it, with potential for further spreading at the point of destination. arboviruses are responsible for a large number of encephalitic diseases characterized by mode of transmission and geographic area. mosquito-borne arboviruses causing encephalitis include eastern and western equine, venezuelan, japanese, and murray hill encephalitides. japanese encephalitis is caused by a mosquito-borne arbovirus found in asia and is associated with rice-growing areas. it is characterized by headache, fever, convulsions, and paralysis, with fatality rates in severe cases as high as %. a currently available vaccine is used routinely in endemic areas (japan, korea, thailand, india, and taiwan) and for persons traveling to infected areas. tick-borne arboviruses causing encephalitis include the powassan virus, which occurs sporadically in the united states and canada. tickborne encephalitis is endemic in eastern europe, scandinavia, and the former soviet union. an epidemic of mosquito-borne encephalitis in new york city in included cases and deaths, due to the west nile fever virus, never before found in the united states. other insect vectors. it affects animals and humans who are in direct contact with the meat or blood of affected animals. the virus causes a generalized illness in humans with encephalitis, hemorrhages, retinitis and retinal hemorrhage leading to partial or total blindness, and death ( - %). it also causes universal abortion in ewes and a high percentage of death in lambs. the normal habitat is in the rift valley of eastern africa (the great syrian-african rift), often spreading to southern africa, depending on climactic conditions. the primary reservoir and vector is the aedes mosquito, and affected animals serve to multiply the virus which is transmitted by other vectors and direct contact with animal fluids to humans. an unusual spread of rvf northward to the sudan and along the aswan dam reservoir to egypt in - caused hundreds of thousands of animal deaths, with , human cases and deaths. rvf appeared again in egypt in . this disease is suspected to be one of the ten plagues of egypt leading to the exodus of the children of israel from egypt during pharaonic-biblical times. in , an outbreak of rvf in kenya, initially thought to be anthrax, with hundreds of cases and dozens of deaths, was related to abnormal rainy season and vector conditions. satellite monitoring of rainfall and vegetation is being used to predict epidemics in kenya and surrounding countries. animal immunization, monitoring, vector control, and reduced contact with infected animals can limit the spread of this disease. arboviruses can also cause hemorrhagic fevers. these are acute febrile illnesses, with extensive hemorrhagic phenomena (internal and external), liver damage, shock, and often high mortality rates. the potential for international transmission is high. yellow fever. yellow fever is an acute viral disease of short duration and varying severity with jaundice. it can progress to liver disease and severe intestinal bleeding. the case fatality rate is < % in endemic areas, but may be as high as % in nonendemic areas and in epidemics. it caused major epidemics in the americas in the past, but was controlled by elimination of the vector, aedes aegypti. a live attenuated vaccine is used in routine immunization endemic areas and recommended for travelers to infected areas. determining the mode of transmission and vector control of yellow fever played a major role in the development of public health (see chapter ). in , the who reported , cases and , deaths from yellow fever globally. dengue hemorrhagic fever. dengue hemorrhagic fever is an acute sudden onset viral disease, with - days of fever, intense headache, myalgia, arthralgia, box . dengue fever and dengue hemorrhagic fever, dengue fever, a severe influenza-like illness, and dengue hemorrhagic fever are closely related conditions caused by four distinct viruses transmitted by aedes aegypti mosquitos. dengue is the world's most important mosquito-borne virus disease. a total of , million people worldwide are at risk of infection. an estimated million cases occur each year, of whom , need to be hospitalized. this is a spreading problem, especially in cities in tropical and subtropical areas. major outbreaks were reported in colombia, cuba, and many other locations in . source: world health organization. . world health report gastrointestinal disturbance, and rash. hemorrhagic phenomena can cause case fatality rates of up to %. epidemics can be explosive, but adequate treatment can greatly reduce the number of deaths. dengue occurs in southeast asia, the pacific islands, australia, west africa, the caribbean, and central and south america. an epidemic in cuba in included more than , cases, and deaths. vector control of the a. aegypti mosquito resulted in control of the disease during the s- s, but reinfestation of mosquitoes led to incresased transmission and epidemics in the pacific islands, caribbean, central and south america in the s and s. outbreaks in vietnam included , cases in , another , cases in , and a similar sized outbreak in . indonesia had over , cases in with deaths, and in over , cases (january-may) with at least deaths. in , epidemics of dengue were reported in fiji, the cook islands, new caledonia, and northern australia. the who estimates , deaths and . million cases worldwide in . monkeys are the main reservoir, and the vector is the a. aegypti mosquito. no vaccine is currently available, and management is by vector control. lassa fever. lassa fever was first isolated in lassa, nigeria, in and is widely distributed in west africa, with , - , cases and deaths annually. it is spread by direct contact with blood, urine, or secretions of infected rodents and by direct person-to-person contact in hospital settings. the disease is characterized by a persistent or spiking fever for - weeks, and may include severe hypotension, shock, and hemorrhaging. the case fatality rate is %. marburg disease. marburg disease is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first seen in marburg, germany, in , following ex-posure to green monkeys. person-to-person spread occurs via blood, secretions, organs, and semen. case fatality rates can be over %. ebola fever. ebola fever is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first found in zaire and sudan in in outbreaks which killed more than persons. it is spread from person to person by the blood, vomitus, urine, stools, and other secretions of sick patients, with a short incubation period. the disease has case fatality rates of up to %. an outbreak of ebola among laboratory monkeys in a medical laboratory near washington, d.c., was contained with no human cases. the reservoir for the virus is thought to be rodents. an outbreak of ebola in may in the town of kikwit, zaire, killed persons out of cases ( % case fatality rate). this outbreak caused international concern that the disease could spread, but it remained localized. another outbreak of ebola virus occurred in gabon in early , with cases, of whom had direct exposure to an infected monkey, the remainder by human-to-human contact, or not established; of the cases died ( %). this disease is considered highly dangerous unless outbreaks are effectively controlled. in zaire, lack of basic sanitary supplies, such as surgical gloves for hospitals, almost ensures that this disease will spread when it recurs. lyme disease is characterized by the presence of a rash, musculoskeletal, neurologic, and cardiovascular symptoms. confirmation is by laboratory investigation. it is the most common vector-borne disease in the united states, with , cases reported between and . it primarily affects children in the - age group and adults aged - . lyme disease is preventable by avoiding contact with ticks, by applying insect repellant, wearing long pants and long sleeves in infected areas, and by the early removal of attached ticks. several u.s. manufacturers produced vaccines which are approved for animal and human use. in the mid s, a mother of two young boys who were recently diagnosed with arthritis in the town of lyme, connecticut, conducted a private investigation among other town residents. she mapped each of the six arthritis cases in the town, cases which had occurred in a short time span among boys living in close proximity. this suggested that this syndrome of "juvenile rheumatoid arthritis" was perhaps connected with the boys playing in the woods. she presented her data to the head of rheumatology at yale medical school in new haven, who investigated this "cluster of a new disease entity." some parents reported that their sons had experienced tick bites and a rash before onset of the arthritis. a tick-borne, spiral shaped bacterium, a spirochete, borrelia burgdorferi, was identified as the organism, and ticks shown to be the vector. cases repond well to antibiotic therapy. in over , cases ( . per , ) were reported from states, an increase from , in and , in . cases were mainly located in the northeast, north central, and mid-atlantic regions. the disease accounts for over % of vector-borne disease in the united states and was the ninth leading reported infection in . lyme disease has been identified in many parts of north america, europe, the former soviet union, china, and japan. a newly licensed vaccine is effective for people exposed to ticks but not general usage. personal hygiene for protection from ticks and environmental modification are important to limit spread of the disease. source: cdc, , mmwr, : - ; and cdc, , mmwr, , no. . lyme disease website http://www.cdc.gov/ncidad/disease/lyme/lyme.htm medically important parasites are animals that live, take nourishment, and thrive in the body of a host, which may or may not harm the host, but never brings benefit. they include those caused by unicellular organisms such as protozoa, which include amoebas (malaria, schistosomiasis, amebiasis, and cryptosporidium), and helminths (worms), which are categorized as nematodes, cestodes, and trematodes. public health continues to face the problems of parasitic diseases in the developing world. increasingly, parasitic diseases are being recognized in industrialized countries. giardiasis and cryptosporidium infections in waterborne and other outbreaks have occurred in the united states. parasitic diseases are among the most common causes of illness and death in the world, e.g., malaria. milder illnesses such as giardiasis and trichomoniasis cause widespread morbidity. intestinal infestations with worms may cause of severe complications, although they commonly cause chronic low-grade symptomatology and iron deficiency anemia. echinococcosis (hydatid cyst disease) is infection with echinococcus granulosus, a small dog tapeworm. the tapeworm forms unilocular (single, noncompartmental) cysts in the host, primarily in the liver and lungs, but they can also grow in the kidney, spleen, central nervous system, or in bones. cysts, which may grow up to cm in size, may be asymptomatic or, if untreated, may cause severe symptoms and even death. this parasite is common where dogs are used with herd grazing animals and also have intimate contact with humans. the middle east, greece, sardinia, north africa, and south america are endemic areas, as are a few areas in the united states and canada. the human dis-ease has been eliminated in cyprus and australia. while the dog is the major host, intermediate hosts include sheep, cattle, pigs, horses, moose, and wolves. preventive measures include education in food and animal contact hygiene, destroying wild and stray dogs, and keeping dogs from the viscera of slaughtered animals. a similar, but multilocular, cystic hydatid disease is widely found in wild animal hosts in areas of the northern hemisphere, including central europe, the former soviet union, japan, alaska, canada, and the north-central united states. another echinococcal disease (echinococcus vogeli) is found in south america, where its natural host is the bush dog and its intermediate host is the rat. the domestic dog also serves as a source of human infection. surgical resection is not always successful, and long-term medical treatment may be required. control is through awareness and hygiene as well as the control of wild animals that come in contact with humans and domestic animals. control may require cooperation between neighboring countries. tapeworm infestation (taeniasis) is common in tropical countries where hygienic standards are low. beef (taenia saginata) and pork (t. solium) tapeworms are common where animals are fed with water or food exposed to human feces. freezing or cooking meat will destroy the tapeworm. fish tapeworm (diphyllobothrium latum) is common in populations living primarily on uncooked fish, such as inuit people. these tapeworms are usually associated with northern climates. toddlers are especially susceptible to dog tapeworm (dipylidium caninum), which is present worldwide, and domestic pets are often the source of oral-fecal transmission of the eggs. the disease is usually asymptomatic. similarly, dwarf tapeworm (hymenolepis nana) is transmitted through oral-fecal contamination from person to person, or via contaminated food or water. rat tapeworm (hymenolepis diminuta) also mostly affects young children. onchocerciasis (fiver blindness) is a disease caused by a parasitic worm, which produces millions of larvae that move through the body causing intense itching, debilitation, and eventually blindness. the disease is spread by a blackfly that transmits the larva from infected to uninfected people. it is primarily located in sub-saharan africa and in latin america, with over million persons at risk. control is by a combination of activities including environmental control by larvicidal sprays to reduce the vector population, protection of potential hosts by protective clothing and insect repellents, and case treatment. a who-initiated program for onchocerciasis control started in is sponsored by four international agencies: the food and agriculture organization (fao), the united nations development program (undp), the world bank, and who. it covers countries in sub-saharan africa, focusing on control of the blackfly by destoying its larvae, mainly via insecticides sprayed from the air. prevalence in was reported by who as over million persons. the program has been successful in protecting some million persons and helping . million infected persons to recover from this disease. who estimates that the program will have prevented , cases of blindness by the year and has freed million hectares of land for resettlement and cultivation. the program cost $ million. this investment is considered by the world bank to have a return of - % in terms of large scale land reuse and improved output of the population. a who program, the african program for onchocerciasis control (apoc), started in , uses a new drug (ivermectin) and selective vector control efforts by spraying. this involves countries in africa, and in a similar program in south america. see website http://www/who.int/ocp and is financed by many donor countries, internation organizations, merck & company, and ngos. dracunculiasis (guinea worm disease) is a parasitic disease of great public health importance in india, pakistan, and central and west africa. it is an infection of the subcutaneous and deeper tissues caused by a large ( cm) nematode, usually affecting the lower extremities and causing pain and disability. the nematode causes a burning blister on the skin when it is ready to release its eggs. after the blister ruptures, the worm discharges larvae whenever the extremity is in water. the eggs are ingested in contaminated water and the larva released migrate through the viscera to locate as adults in the subcutaneous tissue of the leg. incubation is about months. the larva released in water are ingested by minute crustaceans and remain infective for as long as a month. prevention is based on improving the safety of water supplies and by preventing contamination by infected persons. education of persons in endemic areas to stay out of water sources and to filter drinking water reduces transmission. insecticides remove the crustaceans. chlorine also kills the larvae and the crustaceans which prologue larval infectivity. there is no vaccine. treatment is helpful, but not definitive. dracunculiasis was traditionally endemic in a belt from west africa through the middle east to india and central asia. it was successfully eliminated from central asia and iran and has disappeared from the middle east and from some african countries (gambia and guinea). the world health organization has promoted the eradication of dracunculiasis. major progress has been made in this direction. worldwide prevalence is reported to have been reduced from million cases in to million in , , in , and , cases in . eradication was anticipated for the year , and in the who established a commission to monitor and certify eradication in formerly endemic areas. india's reported cases fell from , in to in , and the country was free of transmission in . in , formerly high prevalence countries such as kenya reported no cases in , while chad, senegal, cameroons, yemen, and the central african republic less than cases each. eradication of this disease appears to be imminent. the who eradication program was developed successfully as an independent program with its own direction and field staff, but further progress will require the integration of this program with other basic primary care programs in order to be self-sustaining as an integral part of community health. community-based surveillance systems for this disease are being converted to work for monitoring of other health conditions in the community. schistosomiasis (snail fever or bilharziasis) is a parasitic infection caused by the trematode (blood fluke) and transmitted from person to person via an intermediate host, the snail. it is endemic in countries in africa, south america, the caribbean, and asia. there are an estimated million persons infected worldwide and more than million at risk for the disease. the clinical symptoms include fever, nausea, vomiting, abdominal pain, diarrhea, and hematuria. the organisms schistosoma mansoni and s. japonicum cause intestinal and hepatic symptoms, including diarrhea and abdominal pain. schistosoma haematobium affects the genitourinary tract, causing chronic cystitis, pyelonephritis, with high risk for bladder cancer the ninth most common cause of cancer deaths globally. infection is acquired by skin contact with freshwater containing contaminated snails. the cercariae of the organism penetrate the skin, and in the human host it matures into an adult worm that mates and produces eggs. the eggs are disseminated to other parts of the body from the worm's location in the veins surrounding the bladder or the intestines, and may result in neurological symptoms. eggs may be detected under microscopic examination of urine and stools. sensitive serologic tests are also available. treatment is effective against all three major species of schistosomiasis. eradication of the disease can be achieved with the use of irrigation canals, prevention of contamination of water sources by urine and feces of infected persons, treatment of infected persons, destruction of snails, and health education in affected areas. persons exposed to freshwater lakes, streams, and rivers in endemic areas should be warned of the danger of infection. mass chemotherapy in communities at risk and improved water and sanitation facilities are resulting in improved control of this disease. leishmaniasis causes both cutaneous and visceral disease. the cutaneous form is a chronic ulcer of the skin, called by various names, e.g., rose of jericho, oriental sore, and aleppo boil. it is caused by leishmania tropica, l. brasiliensis, l. mexicana, or the l. donovani complex. this chronic ulcer may last from weeks to more than a year. diagnosis is by biopsy, culture, and serologic tests. the organism multiplies in the gut of sandflies (phlebotomus and lutzomi) and is transmitted to humans, dogs, and rodents through bites. the parasites may remain in the untreated lesion for - months, and the lesion does not heal until the parasites are eliminated. prevention is through limiting exposure to the phlebotomines and reducing the sandfly population by environmental control measures. insecticide use near breeding places and homes has been successful in destroying the vector sandflies in their breeding places. case detection and treatment reduce the incidence of new cases. there is no vaccine, and treatment is with specific antimonials and antibiotics. visceral leishmaniasis (kala azar) is a chronic systemic disease in which the parasite multiplies in the cells of the host's visceral organs. the disease is characterized by fever, the enlargement of the liver and spleen, lymphadenopathy, anemia, leukopenia, and progressive weakness and emaciation. diagnosis is by culture of the organism from biopsy or aspirated material, or by demonstration of intracellular (leishman-donovan) bodies in stained smears from bone marrow, spleen, liver, or blood. kala azar is a rural disease occurring in the indian subcontinent, china, the southern republics of the former u.s.s.r., the middle east, latin america, and sub-saharan africa. it usually occurs as scattered cases among infants, children, and adolescents. transmission is by the bite of the infected sandfly with an incubation period of - months. there is no vaccine, but specific treatment is effective and environmental control measures reduce the disease prevalence. this includes the use of antimalarial insecticides. in localities where the dog population has been reduced, the disease is less prevalent. sleeping sickness. sleeping sickness a disease caused by trypanosoma brucei, transmitted but the tsetse fly, primarily in the african savannahs, affecting cattle and humans. some million persons are at risk in sub-saharan africa. who reported , new cases, a total prevalence of , cases, and , deaths from this disease in . prevention depends on vector control, and effective treatment of human cases. chagas disease is a chronic and incurable vector and blood transfusion borne parasitic disease (trypanosoma cruzi) which causes disability and death. it affects some million persons mainly in latin america, with some , new cases and , deaths occurring annually. about % of affected persons develop severe heart disease. brazil, which accounts for % of the cases prevalent in latin america, achieved elimination of transmission in , after uruguay ( ) and venezuela ( ) and followed by argentina ( ) . elimination of transmission is projected by who by the year . control is difficult, but control measures include reducing the animal host and vector insect population in its habitat by ecological and insectiside measures, education of the population in prevention by clothing, bednets, and repellents, and with chemotherapy for case management. amebiasis. amebiasis is an infection with a protozoan parasite (entamoeba histolytica) which exists as an infective cyst. infestation may be asymptomatic or cause acute, severe diarrhea with blood and mucus, alternating with constipation. amebic colitis can be confused with ulcerative colitis. diagnosis is by microscopic examination of fresh fecal specimens showing trophozoites or cysts. transmission is generally via ingestion of fecal-contaminated food or water containing cysts, or by oral-anal sexual practices. amebiasis is found worldwide. sand filtration of community water supplies removes nearly all cysts. suspect water should be boiled. education regarding hygienic practices with safe food and water handling and disposal of human feces are the basis for control. ascariasis. ascariasis is infestation of the small intestine with the roundworm ascaris lumbricoides, which may appear in the stool, occasionally the nose or mouth, or may be coughed up from lung infestation. the roundworm is very common in tropical countries, where infestation may reach or exceed % of the population. children aged - years are especially susceptible. infestation can cause pulmonary symptoms and frequently contributes to malnutrition, especially iron deficiency anemia. transmission is by ingestion of infective eggs, common among children playing in contaminated areas, or via the ingestion of uncooked products of infected soil. eggs may remain viable in the soil for years. vermox and other treatments are effective. prevention is through education, adequate sanitary facilities for excretion, and improved hygienic practices, especially with food. use of human feces for fertilizer, even after partial treatment, may spread the infestation. mass treatment is indicated in high prevalence communities. pinworm disease or enterobiasis. pinworm disease (oxyuriasis) is common worldwide in all socioeconomic classes; however, it is more widespread when crowded and unsanitary living conditions exist. the enterobius vermicularis infestation of the intestine may be symptomless or may cause severe perianal itching or vulvovaginitis. it primarily affects schoolchildren and preschoolers. more severe complications may occur. adult worms may be seen visually or identified by microscopic examination of stool specimens or perianal swabs. transmission is by the oral-fecal ingestion of eggs. the larvae grow in the small intestine and upper colon. prevention is by educating the public regarding hygiene and adequate sanitary facilities, as well as by treating cases and investigating contacts. treatment is the same as for ascariasis. mass treatment is indicated in high prevalence communities. ectoparasites. ectoparasites include scabies (sarcoptes scabiei), the common bed bug (cimex lectularius), fleas, and lice, including the body louse (pediculus humanis), pubic louse (phthirius pubis), and the head louse (pediculus humanus capitis). their severity ranges from nuisance value to serious public health hazard. head lice are common in schoolchildren worldwide and are mainly a distressing nuisance. the body louse serves as a vector for epidemic typhus, trench fever, and louse-borne relapsing fever. in disaster situations, disinfection and hygienic practices may be essential to prevent epidemic typhus. the flea plays an important role in the spread of the plague by transmitting the organism from the rat to humans. control of rats has reduced the flea population, but during war and disasters, rat and flea populations may thrive. scabies, which is caused by a mite, is common worldwide and is transmitted from person to person. the mite burrows under the skin and causes intense itching. all of these ectoparasites are preventable by proper hygiene and the treatment of cases. the spread of these diseases is rapid and therefore warrants attention in school health and public health policy. legionnelae, a gram-negative group of bacilli, with species and many serogroups. the first documented case was reported in the united states in , and the first disease outbreak was reported in the united states in among participants of a war veterans convention. general malaise, anorexia, myalgia, and headache are followed by fever, cough, abdominal pain, and diarrhea. pneumonia followed by respiratory failure may follow. the case fatality rate can be as high as % of hospitalized cases. a milder, nonpneumonic form of the disease (pontiac fever) is associated with virtually no mortality. the organism is found in water reservoirs and is transmitted through heating, cooling, and air conditioning systems, as well as from tap water, showers, saunas, and jaccuzzi baths. the disease has been reported in australia, canada, south america, europe, israel, and on cruise ships. prevention requires the cleaning of water towers and cooling systems, including whirlpool spas. hyperchlorination of water systems and the replacement of filters is required where cases and/or organisms have been identified. antibiotic treatment with erythromycin is effective. leprosy (hansen's disease) was widely prevalent in europe and mediterranean countries for many centuries, with some , leprosaria in the year . leprosy was largely wiped out during the black death in the fourteenth century, but continued in endemic form until the twentieth century. leprosy is a chronic bacterial infection of the skin, peripheral nerves, and upper airway. in the lepromatous form, there is diffuse infiltration of the skin nodules and macules, usually bilateral and extensive. the tuberculoid form of the disease is characterized by clearly demarcated skin lesions with peripheral nerve involvement. diagnosis is based on clinical examination of the skin and signs of peripheral nerve damage, skin scrapings, and skin biopsy. transmission of the mycobacterium leprae organism is by close contact from person to person, with incubation periods of between months and years (average of - years). rifampicin and other medications make the patient noninfectious in a short time, so that ambulatory treatment is possible. multidrug therapy (mdt) has been shown to be highly effective in combating the disease, with a very low relapse rate. treatment with mdt ensures that the bacillus does not develop drug resistance. mdt is covering % of known cases in , according to who reports, as compared to only % in . the increase has been associated with improved case finding. bcg may be useful in reducing tuberculoid leprosy among contacts. investigation of contacts over years is recommended. the disease is still highly endemic primarily in five countries, india, brazil, indonesia, myanmar, and bangladesh, and is still present in some countries in southeast asia, including the philippines and burma, sub-saharan africa, the middle east (sudan, egypt, iran), and in some parts of latin america (mexico, colombia) with isolated cases in the united states. world prevalence has declined from . million cases in , . million in , to less than million cases in . the world health organization expects to eliminate leprosy as a public health problem by the year , defined as prevalence of less than per , population, or less than , cases. trachoma is currently responsible for million blind persons or % of total blindness in the world. the causative organism, chlamydia trachomatis, is a bacteria which can survive only within a cell. it is spread through contact with eye discharges, usually by flies, or household items (e.g., handkerchiefs, washcloths). trachoma is common in poor rural areas of central america, brazil, africa, parts of asia, and some countries in the eastern mediterranean. the resulting infection leads to conjuncfival scarring and if untreated, to blindness. who estimates there are million cases of active disease in endemic countries. hygiene, vector control, and treatment with antibiotic eye ointments or simple surgery for scarring of eyelids and inturned eyelashes prevent the blindness. a new drug, azithromycin, is effective in curing the disease. the who is promoting a program for the global elimination of trachoma using azithromycin and hygiene education in endemic areas. chlamydia (chlamydia pneumonia) is suspected of playing a role in coronary artery disease by intraarterial infection, with plaque formation and occlusion of the artery by thrombi consisting mainly of platelets. if borne out, this will provide potential for low cost intervention to reduce the burden of the leading worldwide cause of death. sexually transmitted diseases (stds) are widespread internationally with an estimated million new cases per year, with . million new cases, over million total cases, and . million deaths ( ), aids has captured world attention over the past decade. the global burden of stds is enormous (table . ), and the public health and social consequences are devastating in many countries. sexually transmitted diseases, especially in women, may be asymptomatic, so that severe sequelae may occur before patients seek care. infection by one std increases risk of infection by other diseases in this group. syphilis is caused by the spirochete treponema pallidum. after an incubation period of - days (mean - ), primary syphilis develops as a painless ulcer or chancre on the penis, cervix, nose, mouth, or anus, lasting - weeks. the patient may first present with secondary syphilis - weeks (up to weeks) after infection with a general rash and malaise, fever, hair loss, arthritis, and jaundice. these symptoms spontaneously disappear within weeks or up to months later. tertiary syphilis may appear - years after initial infection. complications of tertiary syphilis include catastrophic cardiovascular and central nervous system conditions. early antibiotic treatment is highly effective when given in a large initial dose, but longer term therapy may be needed if treatment is delayed. gonorrhea (gc) is caused by the bacterium neisseria gonorrhoeae. the incubation period is - days. gonorrhea is often associated with concurrent chlamydia infection. in women, gc may be asymptomatic or it may cause vaginal discharge, pain on urination, bleeding on intercourse, or lower abdominal pain. untreated, it can lead to sterility. in men, gc causes urethral discharge and painful urination. treatment with antibiotics ends infectivity, but untreated cases can be infectious for months. drug resistance to penicillin and tetracycline has increased in many countries so that more expensive and often unavailable drugs are necessary for treatment. prevention of gonococcal eye infection in newborns is based on routine use of antibiotic ointments in the eyes of newborns. chancroid. chancroid is caused by haemophilus ducreyi. in women chancroids may cause a painful, irregular ulcer near the vagina, resulting in pain on in-tercourse, urination, and defection, but it may be asymptomatic. in men it causes a painful, irregular ulcer on the penis. the incubation period is usually - days, but may be up to days. an individual is infectious as long as there are ulcers, usually - months. treatment is by erythromycin or azithromycin. herpes simplex. herpes simplex is caused by herpes simplex virus types and and has an incubation period of - days. genital herpes causes painful blisters around the mouth, vagina, penis, or anus. the genital lesions are infectious for - days. herpes may lead to central nervous system meningoencephalitis infection. it can be transmitted to newborns during vaginal delivery, causing infection, encephalitis, and death. cesarian delivery is therefore necessary when a mother is infected. anti-viral drugs are used in treatment, orally, topically, or intravenously. chlamydia. chlamydia is caused by chlamydia trachomatis. in women, it is usually asymptomatic but may cause vaginal discharge, spotting, pain on urination, lower abdominal pain, and pelvic inflammatory disease (pid). in newborns, chlamydia may cause eye and respiratory infections. in men, chlamydia causes urethral discharge and pain on urination. the incubation period is - days and the infectious period is unknown. treatment for chlamydia is doxycycline, azithromycin, or erythromycin. chlamydia infection, not necessarily venereal in transmission, may be transmitted to newborns of infected mothers. chlamydia pneumoniae, presently under investigation as a possible cause or contributor to coronary heart disease, and is widespread in poor hygenic conditions. trichomoniasis. trichomoniasis is caused by trichomonas vaginalis. the incubation period is - days (mean = ). in women, trichomoniasis may be asymptomatic or may cause a frothy vaginal discharge with foul odor, and painful urination and intercourse. in men, the disease is usually mild, causing pain on urination. treatment is by metronidazole taken orally. without treatment, the disease may persist and remain infectious for years. (hpv). it is a sporadic disease which may be associated with cervical neoplasia and cancer of the cervix. hpv includes many types associated with a variety of conditons. the search for a hpv vaccine to prevent cancer of the cervix looks promising. in areas where a full range of diagnostic services is lacking, a "syndromic approach" is recommended for the control of stds. the diagnosis is based on a group of symptoms and treatment on a protocol addressing all the diseases that could possibly cause those symptoms, without expensive laboratory tests and repeated visits. early treatment without laboratory confirmation helps to cure persons who might not return for follow-up, or may place them in a noninfective stage so that even without follow-up they will not transmit the disease. std incidence between and is shown in table . , with decline overall except around , with subsequent further fall in incidence. screening in prenatal and family planning clinics, prison medical services, and selected years - disease syphilis ( [ ] [ ] [ ] [ ] [ ] [ ] and subsequent decline by more than % in reported cases includes all three stages of the disease as well as congential syphilis. rates are cases per , population, rounded. in clinics serving prostitutes, homosexuals, or other potential risk groups will detect subclinical cases of various stds. treatment can be carried out cheaply and immediately. for instance, the screening test for syphilis costs $ . and the treatment with benzathine penicillin injection costs about $ . in . partner notification is a controversial issue, but may be needed to identify contacts who may be the source of transmission to others. control of stds through a syndrome approaach based on primary care providers is being promoted by who. health education directed at high risk target groups is essential. providing easy and cost-free access to acceptable, nonthreatening treatment is vital in promoting the early treatment of cases and thereby reducing the risk of transmission. promoting prevention through the use of condoms and/or monogamy requires long-term educational efforts that are now fostered by the hiv/aids pandemic. increased use of condoms for hiv prevention is associated with reduced risk of other stds. training medical care providers in std awareness should be stressed in undergraduate and continuing educational efforts including personal protection as care givers. human immunodeficiency virus (hiv) is a retrovirus that infects various cells of the immune system, and also affects the central nervous system. two types have been identified: hiv , worldwide in distribution, and the less pathogenic hiv , found mainly in west africa. hiv is transmitted by sexual contact, exposure to blood and blood products, perinatally, and via breast milk. the period of communicability is unknown, but studies indicate that infectiousness is high, both during the initial period after infection and later in the disease. antibodies to hiv usually appear within - months. within several weeks to months of the infection, many persons develop an acute self-limited flulike syndrome. they may then be free of any signs or symptoms for months to more than years. onset of illness is usually insidious with nonspecific symptoms, including sweats, diarrhea, weight loss, and fatigue. aids represents the later clinical stage of hiv infection. according to the revised cdc case definition ( ), aids involves any one or more of the following: low cd count, severe systematic symptoms, opportunistic infections such as pneumocystis pneumonia or tb, aggressive cancers such as kaposi's sarcoma or lymphoma, and/or neurological manifestations, including dementia and neuropathy. the who case definition is more clinically oriented, relying less on often unavailable laboratory diagnoses for indicator diseases. aids was first recognized clinically in in los angeles and new york. by mid- it was considered an epidemic in those and other u.s. cities. it was primarily seen among homosexual men and recipients of blood products. after initial errors, testing of blood and blood products became standard and has subsequently closed off this method of transmission. transmission has changed markedly since the initial onslaught of the disease, with needle sharing among intravenous drug users, heterosexual, and maternal-fetal transmission becoming major factors. comorbidity with other stds apparently increases hiv infectivity and may have helped to convert the epidemiology to a greater degree of heterosexual transmission. the disease grew exponentially in the united states (table . ), but incidence of new cases nas declined since . aids has become a major public health problem in most developed and developing countries, reaching catastrophic proportions in some sub-saharan african countries affecting up to % of the population. hiv-related deaths were the eighth leading cause of all deaths in in the u.s., the leading cause among men aged - years of age, and the fourth leading cause for women in this age group. by , aids had been diagnosed in , persons and , had died. it is estimated that up to million persons are hiv infected in the united states. globally, deaths from aids totalled . million in , with an estimated . million person having died from this pandemic up to . in , an estimated . million person were hiv infected with . million new infection in . the declining incidence of new cases in the industrialized countries may be the result of greater awareness of the disease and methods of prevention of transmission. improving early diagnosis and access to care, especially the combined therapy programs that are very effective in delaying onset of symptoms, are important parts of public health management of the aids crisis. until an effective vaccine is available, preventive reliance will continue to be on behavior risk-reduction and other prevention strategies such as needle and condom distribution among high risk population groups. throughout the world, hiv continues to spread rapidly, especially in poor countries in africa, asia, and south and central america. the united nations reports that million persons are living with hiv/aids, % of them in developing countries, where transmission is % by heterosexual contact. every day, more than persons are infected, including children. in thailand, person in is now infected. in sub-saharan africa person in is infected, and in some cities as many as person in carries the virus. estimations of new infections per year in sub-saharan africa range from to million persons, while in asia the range is from . to . million new infected persons per year. lessons are still being learned from the aids pandemic. the explosive spread of this infection, from an estimated , people in to an anticipated million persons hiv infected, shows that the world is still vulnerable to pandemics of "new" infectious diseases. enormous movements of tourists, business people, truck drivers, migrants, soldiers, and refugees promote the spread of such diseases. widespread sexual exchange, traffic in blood products, and illicit drug use all promote the international potential for pandemics. war and massive refugee situations promote rape and prostitution, worsening the aids situation in some settings in africa. hiv has arrived in almost every country. however, there is the somewhat hopeful indication that the rate of increase, has slowed in the united states. this may be an indication either of higher levels of self-protective behavior, or that the most susceptible population groups have already been affected and the spread into the general population is at a slower rate. it is also possible that this may yet prove to be only a lull in the storm, as heterosexual contact becomes a more important mode of transmission. the eleventh international conference on aids, held in vancouver, canada, in july , reported signs that combinations of several drugs from among a number of antiretroviral medications are showing promise to suppress the aids virus in infected people. at a current annual price of $ , - , per patient, these sums well beyond the capacity of most developing countries. development of methods of measuring the hiv viral load have allowed for better evaluation of potential therapies and monitoring of patients receiving therapy. in developed countries, transmission by blood products has been largely controlled by screening tests; transmission among homosexuals has been reduced by safe sex practices; transmission to newborns has been reduced by recent therapeutic advances. safe sex practices and condom use may have helped in reducing heterosexual transmission. further advances in therapy and prevention with a vaccine are expected over the next decade. the hiv/aids pandemic is one of the great challenges to public health for the st century due to its complexity, its international spread, its sexual and other modes of transmission, its devastating and costly clinical effects, and its impact on parallel diseases such as tuberculosis, respiratory infections, and cancer. the cost of care for the aids patient can be very high. needed programs include home care and community health workers to improve nutrition and self-care, and mutual help among hiv carriers and aids patients. the ethical issues associated with aids are also complex regarding screening of pregnant women, newborns, partner notification, reporting, and contact tracing, as well as financing the cost of care. diarrheal diseases are caused by a wide variety of bacteria, parasites, and viruses (table . ) infecting the intestinal tract and causing secretion of fluids and dis- solved salts into the gut with mild to severe or fatal complications. in developing countries, diarrheal diseases account for half of all morbidity and a quarter of all mortality. diarrhea itself does not cause death, but the dehydration resulting from fluid and electrolyte loss is one of the most common causes of death in children worldwide. deaths from dehydration can be prevented by use of oral rehydration therapy (ort), an inexpensive and simple method of intervention easily used by a nonmedical primary care worker and by the mother of the child as a home intervention. in , diarrheal diseases were the cause of almost million child deaths, but by this had declined to . million, largely under the impact of increased use of ort. diarrheal diseases are transmitted by water, food, and directly from person to person via oral-fecal contamination. diarrheal diseases occur in epidemics in situations of food poisoning or contaminated water sources, but can also be present at high levels when common source contamination is not found. contamination of drinking water by sewage and poor management of water supplies are also major causes of diarrheal disease. the use of sewage for the irrigation of vegetables is a common cause of diarrheal disease in many areas. salmonella are a group of bacterial organisms causing acute gastroenteritis, associated with generalized illness including headache, fever, abdominal pains, and dehydration. there are over serotypes of salmonella, many of which are pathogenic in humans, the most common of which are salmonella typhimurium, s. enteritidis, and s. typhi. transmission is by ingestion of the organisms in food, derived from fecal material from animal or human contamination. common sources include raw or uncooked eggs, raw milk, meat, poultry and its products, as well as pet turtles or chicks. fecal-oral transmission from person to person is common. prevention is in safe animal and food handling, refrigeration, sanitary preparation and storage, protection against rodent and insect contamination, and the use of sterile techniques during patient care. antibiotics may not eliminate the carrier state and may produce resistant strains. shigella are a group of bacteria that are pathogenic in man, with four groups: type a = shigella dysenteriae, type b = s. flexneri, type c = s. boydii, and type d = s. sonnei. types a, b, and c are each further divided into a total of serotypes. shigella are transmitted by direct or indirect fecal-oral methods from a patient or carrier, and illness follows ingestion of even a few organisms. water and milk transmission occurs as a result of contamination. flies can transmit the organism, and in nonrefrigerated foods the organism may multiply to an infectious dose. control is in hygienic practices and in the safe handling of water and food. escheria eoli e. coli are common fecal contaminants of inadequately prepared and cooked food. particularly virulent strains such as o :h can cause explosive outbreaks of severe (enterohemmorhagic) diarrhoeal disease with a hemolytic-uremic syndrome and death, as occurred in japan in with cases and deaths due to a foodborne epidemic. other milder strains cause travellers diarrhoea and nursery infections. inadequately cooked hamburger, unpasturized milk, and other food vectors are discussed under food safety in chapter . cholera is an acute bacterial enteric disease caused by vibrio cholerae, with sudden onset, profuse painless watery stools, occasional vomiting, and, if untreated, rapid dehydration, and circulatory collapse, and death. asymptomatic infection or carrier status, and mild cases are common. in severe, untreated cases, mortality is over %, but with adequate treatment, mortality is under %. diagnosis is based on clinical signs, epidemiologic, serologic and bacteriologic confirmation by culture. the two types of cholera are the classic and el tor (with inaba and ogawa serotypes). in , a large scale epidemic of cholera spread through much of south america. it was imported via a chinese freighter, whose sewage contaminated shellfish in lima harbor in peru (box . ). the south american cholera epidemic has caused hundreds of thousands of cases and thousands of deaths since . prevention requires sanitation, particularly the chlorination of drinking water, prohibiting the use of raw sewage for the irrigation of vegetable crops, and high standards of community, food, and personal hygiene. treatment is prompt fluid therapy with electrolytes in large volume to replace all fluid loss. oral rehydration should be accomplished using standard ort. tetracycline shortens the duration of the disease, and chemoprophylaxis for contacts following stool samples may help in reducing its spread. a vaccine is available but is of no value in the prevention of outbreaks. viral gastroenteritis can occur in sporadic or epidemic forms, in infants, children, or adults. some viruses, such as the rotaviruses and enteric adenoviruses, af- in the s, peruvian officials stopped the chlorination of community water supplies because of concern over possible carcinogenic effects of trihalomethanes, a view encouraged by officials of the u.s. environmental protection agency (epa) and the u.s. public health service. in january , a chinese freighter arrived in lima, peru, and dumped bilge (sewage) in the harbor, apparently contaminating local shellfish. consumption of raw shellfish is a popular local delicacy (ceviche) and associated with cases of cholera seen in local hospitals. contamination of local water supplies from sewage resulted in the geometric increase in cases, and by the end of the pan american health organization (paho) reported an epidemic of , cases and deaths. the epidemic spread to countries, and in there were a further , cases and deaths spreading over much of south america, continuing in . in the united states, cases of cholera were reported in ; of these, cases and death were among passengers of an airplane flying from south america to los angeles in which contaminated seafood was served. in , cases of cholera were reported in the united states which were unrelated to international travel. these occurred mostly among persons consuming shellfish from the gulf coast with a strain of cholera similar to the south american strain, also possibly introduced in ship ballast. cholera organisms are reported in harbor waters in other parts of the united states (promed, , promed, . fect mainly infants and young children, and may be severe enough to cause hospitalization for dehydration. others such as norwalk and norwalk-like viruses affect older children and adults in self-limited acute gastroenteritis in family, institution, or community outbreaks. rotaviruses cause acute gastroenteritis in infants and young children, with fever and vomiting, followed by watery diarrhea and occasionally severe dehydration and death if not adequately treated. diagnosis is by examination of stool or rectal swabs with commercial immunologic kits. in both developed and developing countries, rotavirus is the cause of about one-third of all hospitalized cases for diarrheal diseases in infants and children up to age . most children in developing countries experience this disease by the age of years, with the majority of cases between and months. in developing countries, rotaviruses are estimated to cause over , deaths per year. the virus is found in temperate climates in the cooler months and in tropical countries throughout the year. breastfeeding does not prevent the disease but may reduce its severity. oral rehydration therapy is the key treatment. a live attenuated vaccine was approved by the fda in and adopted in the u.s. recommended routine vaccination programs for infants. adenoviruses. adenoviruses, norwalk, and a variety of other viruses (including astrovirus, calcivirus, and other groups) cause sporadic acute gastroenteritis worldwide, mostly in outbreaks. spread is by the oral-fecal route, often in hospital or other communal settings, with secondary spread among family contacts. food-borne and waterborne transmission are both likely. these can be a serious problem in disaster situations. no vaccines are available. management is with fluid replacement and hygienic measures to prevent secondary spread. giardiasis. giardiasis (caused by giardia lamblia) is a protozoan parasitic infection of the upper small intestine, usually asymptomatic, but sometimes associated with chronic diarrhea, abdominal cramps, bloating, frequent loose greasy stools, fatigue, and weight loss. malabsorption of fats and vitamins may lead to malnutrition. diagnosis is by the presence of cysts or other forms of the organism in stools, duodenal fluid, or in intestinal mucosa from a biopsy. this disease is prevalent worldwide and affects mostly children. it is spread in areas of poor sanitation and in preschool settings and swimming pools, and is of increasing importance as a secondary infection among immunocompromised patients, especially those with aids. waterborne giardia was recognized as a serious problem in the united states in the s and s, since the protozoa is not readily inactivated by chlorine, but requires adequate filtration before chlorination. person-to-person transmission in day-care centers is common, as is transmission by unfiltered stream or lake water where contamination by human or animal feces is to be expected. an asymptomatic carrier state is common. prevention relies on careful hygiene in settings such as day-care centers, filtration of public water supplies and the boiling of water in emergency situations. cryptosporidium. cryptosporidium parvum is a parasitic infection of the gastrointestinal tract in man, small and large mammals and vertebrates. infection may be asymptomatic or cause a profuse, watery diarrhea, abdominal cramps, general malaise, fever, anorexia, nausea, and vomiting. in immunosuppressed patients, such as persons with aids, it can be a serious problem. the disease is most common in children under years of age and those in close contact with them, as well as in homosexual men. diagnosis is by identification of the cryptosporidium or-ganism cysts in stools. the disease is present worldwide. in europe and the united states, the organism has been found in < to . % of individuals sampled. spread is common by person-to-person contact by fecal-oral contamination, especially in such settings as day-care centers. raw milk and waterborne outbreaks have also been identified in recent years. a large waterborne disease outbreak due to cryptosporidium occurred in milwaukee in described in chapter . management is by rehydration and prevention is by careful hygiene in food and water safety. helicobacter pylori. helicobacter pylori, first identified in , is a bacterium causally linked to duodenal ulcers and gastritis, contributing to high rates of gastric cancer (chapter ). it is an important example of the link between infection and chronic disease. this has enormous implications for prevention of cancer of the stomach, chronic peptic ulcers and large-scale use of hospitals and other medical resources (see chapter ). the control of diarrheal diseases requires a comprehensive program involving a wide range of activities, including good management of food and water supplies, education in hygiene, and, particularly where morbidity and mortality are high, education in the use of oral rehydration therapy (ort). oral rehydration therapy (ort) is considered by unicef and who to have resulted in the saving of million lives each year in the s. proper management of an episode of diarrhea by ort (table . ), along with continued feeding, not only saves the child from dehydration and immediate death, but also contributes to early restoration of nutritional adequacy, sparing the child the prolonged effects of malnutrition. the world summit for children (wsc) in called for a reduction in child deaths from diarrheal diseases by one-third and malnutrition by one-half, with em- phasis on the widest possible availability, education for, and use of ort. this requires a programmatic approach. public health leadership must train primary care doctors, pediatricians, pharmacists, drug manufacturers, and primary care health workers of all kinds in ort principles and usage. they must be backed by the widest possible publicity to raise awareness among parents. oral rehydration therapy is an important public health modality in developed countries as well as in developing countries. diarrhoeal disease may not cause death as frequently in developed countries, but it is still a significant factor in infant and child health and, even under the most optimal conditions, can cause setbacks in the nutritional state and physical development of a child. use of ort does not prevent the disease (i.e., it is not a primary prevention), but it is excellent in secondary prevention, by preventing complications from diarrhoea, and should be available in every home for symptomatic treatment of diarrheal diseases. an adaptation of ort has found its place in popular culture in the united states. a form of ort, marketed as "sports drinks," is used in sports where athletes lose large quantifies of water and salts in sweat and insensible loss from the respiratory tract. the wider application of the principles of ort for use in adults in dry hot climates and in adults under severe physical exertion with inadequate fluid/salt intake situations requires further exploration. management of diarrheal diseases should be part of a wider approach to child nutrition. the child who goes through an episode of diarrheal disease may have a faltering in growth and development. supportive measures may be needed following the episode as well as during it. this involves providing primary care services that are attuned to monitoring individual infant and child growth. growth monitoring surveillance is important to assess the health status of the individual child and the child population. supplementation of infant feeding with vitamins a and d, and iron to prevent anemia are important for routine infant and child care, and more so for conditions affecting total nutrition such as a diarrheal disease. in the developing world, respiratory infections account for over one-quarter of all deaths and illnesses in children. as diarrheal disease deaths are reduced, the major cause of death among infants in developing countries is becoming acute respiratory infections (aris). in industrialized countries, aris are important for their potentially devastating effects on the elderly and chronically ill. they are also the major cause of morbidity in infants in developed countries, causing much anxiety to parents even in areas with good living conditions. cigarette smoking, chronic bronchitis, poorly controlled diabetes or congestive heart failure, and chronic liver and kidney disease increase susceptibility to aris. aris place a heavy burden on health care systems and individual families. improved methods of management of such chronic diseases are needed to reduce the associated toll of morbidity, mortality, and the considerable expenses of health care. acute respiratory infections are due to a broad range of viral and, to a lesser extent, bacterial infections. it is the latter which can progress to pneumonia with mortality rates of - %. acute viral respiratory diseases include those affecting the upper respiratory tract, such as acute viral rhinitis, pharyngitis, and laryngitis, as well as those affecting the lower respiratory tract, tracheobronchitis, bronchitis, bronchiolitis, and pneumonia. aris are frequently associated with vaccine-preventable diseases, including measles, varicella, and influenza. they are caused by a large number of viruses, producing a wide spectrum of acute respiratory illness. some organisms affect any part of the respiratory tract, while others affect specific parts and all predispose to bacterial secondary infection. while children and the elderly are especially susceptible to morbidity and mortality from acute respiratory disease, the vast numbers of respiratory illnesses among adults cause large-scale economic loss from work absence. bacterial agents causing upper respiratory tract infection include group a streptococcus, mycoplasma pneumonia, pertussis, and parapertussis. pneumonia or acute bacterial infection of the lower respiratory tract and lung tissue may be due to pneumococcal infection with streptococcus pneumoniae. there are known types of this organism, distinguished by capsule characteristics; account for % of pneumococcal infections in the united states. an excellent polyvalent vaccine based on these types is available for high risk groups such as the elderly, immunodeficient patients, and persons with chronic heart, lung, liver, blood disorders, or diabetes. opportunistic infections attack the chronically ill, especially those with compromised immune suystems, often with life-threatening aris. mycoplasma (primary atypical pneumonia) is a lower respiratory tract infection which sometimes progresses to pneumonia. tb and pneumonocytis carynia are especially problematic for aids patients. other organisms causing pneumonias include chlamydia pneumoniae, h. influenza, klebsiella pneumonia, escherichia coli, staphylococcus, rickettsia (q fever), and legionella. parasitic infestation of lungs may occur with nematodes (e.g., ascariasis). fungal infections of the lung may be caused by aspergillosis, histoplasmosis, and coccidiomycosis, often as a complication of antibiotic therapy. access to primary care and early institution of treatment are vital to control excess mortality from aris. in developed countries, aris as contributors to infant deaths are largely a problem in minority and deprived population groups. because these groups contribute disproportionately to childhood mortality, infant mortality reduction has been slower in countries such as the united states and russia than in other industrialized countries. the continuing gap in mortality rates between white and black children in the united states can, to a large extent, be attributed to aris and less access to organized primary care. children are brought to emergency rooms for care when the disease process is already advanced and more dangerous than had it been attended to professionally earlier in the process. many field trials of ari prevention programs have been proved successful involving parent education and training of primary care workers in early assessment and, if necessary, initiation of treatment. this needs field testing in multiple settings. reliance on vaccines to prevent respiratory infectious diseases is not currently feasible. aris are caused by a very wide spectrum of viruses, and the development of vaccines in this field has been slow and limited. the vaccine for pneumococcal pneumonia has been an important breakthrough, but it is still inadequately utilized by the chronically ill because of its limitations, costs, and lack of sufficient awareness, and it is too expensive for developing countries. improvements in bacterial and viral vaccine development will potentially help to reduce the burden of aris. a programmatic approach with clinical guidelines and education of family and care givers is currently the only feasible way to reduce the still enormous morbidity and mortality from aris on the young and the elderly. the success of sanitation vaccines and antibiotics led many to assume that all infectious diseases would sooner or later succumb to public health and medical technology. unfortunately, this is a premature and even dangerous assumption. despite the longstanding availability of an effective and inexpensive vaccine, the persistence of measles as a major killer of million children per year represents a failure in effective use of both the vaccine and the health system. the resurgence of tb and malaria have led to new strategies, such as managed or directly observed care, with community health workers to assure compliance needed to render the patient noninfectious to others and to reduce the pool of carriers of the disease. current successes in reducing poliomyelitis, dracunculiasis, onchocerciasis, and other diseases to the point of eradication has raised hopes for similar success in other fields. but there are many infectious diseases of importance in developed and developing countries where existing technologies are not fully utilized. oral rehydration therapy (ort) is one of the most cost-effective methods of preventing excess mortality from ordinary diarrheal diseases, and yet is not used on sufficient scale. biases in the financing and management of medical insurance programs can result in underutilization of available effective vaccines. hospital-based infections cause large-scale increases in lengths of stay and expenditures, although application of epidemiologic investigation and improved quality in hospital practices could reduce this burden. control of the spread of aids using combined medical therapies is not financially or logistically possible in many countries, but education for "safe sex" is effective. community health worker programs can greatly enhance tuberculosis, malaria, and std control, or in aids care, promote prevention and appropriate treatment. in the industrialized and mid-level developing countries, epidemiologic and demographic shifts have created new challenges in infectious disease control. prevention and early treatment of infectious disease among the chronically ill and the elderly is not only a medical issue, it is also an economic one. patients with chronic obstructive lung disease (copd), chronic liver or kidney disease, or congestive heart failure are at high risk of developing an infectious disease followed by prolonged hospitalization. public health has addressed, and will continue to stress the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. the eradication of smallpox paved the way for the eradication of poliomyelitis, and perhaps measles, in the foreseeable future. new vaccines are showing the capacity to reduce important morbidity from rubella syndrome, mumps, meningitis, and hepatitis. other new vaccines on the horizon will continue the immunologic revolution into the twenty-first century. as the triumphs of control or elimination of infectious diseases of children continue, the scourge of hiv infection continues with distressingly slow progess an effective vaccine or cure for the disease it engenders. partly as a result of the hiv/ aids, tb staged a comeback in many countries where it was thought to be merely a residual problem. at the same time an old/new method of intervention using directly observed short-term therapy has shown great success in controlling the tb epidemic. the resurgence of tb is more dangerous in that mdrtb has become a widespread problem. this issue highlights the difficulty of keeping ahead of drug resistance in the search for new generations of antibiotics, posing a difficult challenge for the pharmaceutical industry, basic scientists as well as public health workers. the burden of infectious diseases has receded as the predominant public health problem in the developed countries but remains large in the developing countries. with increases in longevity and increased importance of chronic disease in the health status of the industrial and mid-level developing nations, the effects of infectious disease on the care of the elderly and chronically ill is of great importance in the new public health. long-term management of chronic disease needs to address the care of vulnerable groups, promoting the use of existing vaccines and antibiotics. most important is the development of health systems that provide close monitoring of groups at special risk for infectious disease, especially patients with chronic diseases, the immunocompromised, and the elderly. the combination of traditional public health with direct medical care needed for effective control and eradication of communicable diseases is an essential element of the new public health. the challenge is to apply a comprehensive approach and management of resources to define and reach achievable targets in communicable disease control. access to e-mail and the internet are vital to current practice of public health and nowhere is this more important than in communicable diseases. there are many such information sites and these will undoubtedly expand in the coming years. several sites are given as examples. the internet has great practical implications for keeping up to date with rapidly occurring events in this field. outstanding encyclopedia database on infectious diseases (available via mdcassoc@ix.netcom.com at reduced price for promed users, and free to sub-saharan african sites) promed is an excellent, free report on current events in communicable diseases internationally; join via owner-promed @usa recommended readings centers for disease control. . update: international task force for disease eradication addressing emerging infectious disease threats: a prevention strategy for the united states. executive summary update: trends in aids incidence--united states one thousand days until the target date for global poliomyelitis eradication tuberculosis morbidity--united states measles--united states, . morbidity and mortality weekly report national adult immunization awareness week--october - , recommended readings ; and influenza and pneumococcal vaccination levels among adults aged --- years impact of the sequential ipv/opv schedule on vaccination cover-agemunited states advances in global measles control and elimination: summary of the international meeting recommended childhood immunization schedulemunited states impact of vaccines universally recommended for childrenmunited states progress toward global poliomyelitis eradication global disease elimination and eradication as public health strategies childhood immunizations rotavirus vaccines: who position paper. weekly epidemiologic record infectious diseases of humans: dynamic and control vaccines and world health: science, policy, and practice control of communicable diseases manual jawetz, melnick and adelberg's medical microbiology, twenty-first edition preventive medicine and public health, second edition efficacy of bcg vaccine in the prevention of tuberculosis. meta-analysis of the published literature manson's tropical diseases vaccination and world health principles and practice oflnfectious diseases immunization of adolescents: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the combination vaccines for childhood immunization: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the poliomyelitis prevention: revised recommendations for use of inactivated and live oral poliovirus vaccines diphtheria outbreakmrussian federation rubella and congenital rubella syndrome~united states compendium of animal rabies control, : national association of state public health veterinarians progress toward elimination of haemophilus influenzae type b disease among infants and children in the united states tetanus surveillance~united states, - recommendations and reports--vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of measles: recommendations of the advisory committee on immunization practices national, state and urban area vaccination coverage levels among children aged - months~united sates varicella related deaths among children--united states progress toward global poliomyelitis eradication ten great public health achievements--united states a ten-year experience in control of poliomyelitis through a combination of live and killed vaccines in two developing areas measles control in developing and developed countries: the case for a two-dose policy integration of vitamin a supplementation with immunization. weekly epidemiological record update cholera--western hemisphere, . morbidity and mortality weekly report isolation of vibrio cholerae o from oystersmmobile bay, - estimates of future global tuberculosis morbidity and mortality arbovirus disease--united states ~:~ other communicable diseases update: outbreak of legionnaire's disease associated with a cruise ship rift valley fever--egypt the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices update: trends in aids incidence--united states case definition for infectious conditions under public health surveillance guidelines for treatment of sexually transmitted diseases primary and secondary syphilis--united states global tuberculosis incidence and mortality during the th century pandemic: need for surveillance and research escherichia coli o :h diarrhoea in the united states: clinical and epidemiologic features the state of the world's children the rational use of drugs in the management of acute diarrhoea in children world health organization. . the malaria situation in aids: images of the epidemic. geneva: who. world health organization progress toward the elimination of leprosy as a public health problem the world health report : fighting disease, fostering development the world health report health for all in the twenty-first century. eb / . geneva: who. world health organization. . the world health report : life in the twenty-first century: a vision for all world health organization. . the world health report : making a difference key: cord- -k imddzr authors: siegel, jane d.; rhinehart, emily; jackson, marguerite; chiarello, linda title: guideline for isolation precautions: preventing transmission of infectious agents in health care settings date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: k imddzr nan . clinical syndromes or conditions warranting additional empiric transmission-based precautions pending confirmation of diagnosis table . infection control considerations for highpriority (cdc category a) diseases that may result from bioterrorist attacks or are considered bioterrorist threats table . recommendations for application of standard precautions for the care of all patients in all health care settings table . components of a protective environment . the transition of health care delivery from primarily acute care hospitals to other health care settings (eg, home care, ambulatory care, freestanding specialty care sites, long-term care) created a need for recommendations that can be applied in all health care settings using common principles of infection control practice, yet can be modified to reflect setting-specific needs. accordingly, the revised guideline addresses the spectrum of health care delivery settings. furthermore, the term ''nosocomial infections'' is replaced by ''health care-associated infections'' (hais), to reflect the changing patterns in health care delivery and difficulty in determining the geographic site of exposure to an infectious agent and/ or acquisition of infection. . the emergence of new pathogens (eg, severe acute respiratory syndrome coronavirus [sars-cov] associated with sars avian influenza in humans), renewed concern for evolving known pathogens (eg, clostridium difficile, noroviruses, communityassociated methicillin-resistant staphylococcus aureus [ca-mrsa]), development of new therapies (eg, gene therapy), and increasing concern for the threat of bioweapons attacks, necessitates addressing a broader scope of issues than in previous isolation guidelines. . the successful experience with standard precautions, first recommended in the guideline, has led to a reaffirmation of this approach as the foundation for preventing transmission of infectious agents in all health care settings. new additions to the recommendations for standard precautions are respiratory hygiene/cough etiquette and safe injection practices, including the use of a mask when performing certain highrisk, prolonged procedures involving spinal canal punctures (eg, myelography, epidural anesthesia). the need for a recommendation for respiratory hygiene/cough etiquette grew out of observations during the sars outbreaks, when failure to implement simple source control measures with patients, visitors, and health care workers (hcws) with respiratory symptoms may have contributed to sars-cov transmission. the recommended practices have a strong evidence base. the continued occurrence of outbreaks of hepatitis b and hepatitis c viruses in ambulatory settings indicated a need to reiterate safe injection practice recommendations as part of standard precautions. the addition of a mask for certain spinal injections grew from recent evidence of an associated risk for developing meningitis caused by respiratory flora. . the accumulated evidence that environmental controls decrease the risk of life-threatening fungal infections in the most severely immunocompromised patients (ie, those undergoing allogeneic hematopoietic stem cell transplantation [hsct] ) led to the update on the components of the protective environment (pe). . evidence that organizational characteristics (eg, nurse staffing levels and composition, establishment of a safety culture) influence hcws' adherence to recommended infection control practices, and thus are important factors in preventing transmission of infectious agents, led to a new emphasis and recommendations for administrative involvement in the development and support of infection control programs. . continued increase in the incidence of hais caused by multidrug-resistant organisms (mdros) in all health care settings and the expanded body of knowledge concerning prevention of transmission of mdros created a need for more specific recommendations for surveillance and control of these pathogens that would be practical and effective in various types of health care settings. this document is intended for use by infection control staff, health care epidemiologists, health care administrators, nurses, other health care providers, and persons responsible for developing, implementing, and evaluating infection control programs for health care settings across the continuum of care. the reader is referred to other guidelines and websites for more detailed information and for recommendations concerning specialized infection control problems. part i reviews the relevant scientific literature that supports the recommended prevention and control practices. as in the guideline, the modes and factors that influence transmission risks are described in detail. new to the section on transmission are discussions of bioaerosols and of how droplet and airborne transmission may contribute to infection transmission. this became a concern during the sars outbreaks of , when transmission associated with aerosol-generating procedures was observed. also new is a definition of ''epidemiologically important organisms'' that was developed to assist in the identification of clusters of infections that require investigation (ie multidrug-resistant organisms, c difficile). several other pathogens of special infection control interest (ie, norovirus, sars, centers for disease control and prevention [cdc] category a bioterrorist agents, prions, monkeypox, and the hemorrhagic fever viruses) also are discussed, to present new information and infection control lessons learned from experience with these agents. this section of the guideline also presents information on infection risks associated with specific health care settings and patient populations. part ii updates information on the basic principles of hand hygiene, barrier precautions, safe work practices, and isolation practices that were included in previous guidelines. however, new to this guideline is important information on health care system components that influence transmission risks, including those components under the influence of health care administrators. an important administrative priority that is described is the need for appropriate infection control staffing to meet the ever-expanding role of infection control professionals in the complex modern health care system. evidence presented also demonstrates another administrative concern: the importance of nurse staffing levels, including ensuring numbers of appropriately trained nurses in intensive care units (icus) for preventing hais. the role of the clinical microbiology laboratory in supporting infection control is described, to emphasize the need for this service in health care facilities. other factors that influence transmission risks are discussed, including the adherence of hcws to recommended infection control practices, organizational safety culture or climate, and education and training. discussed for the first time in an isolation guideline is surveillance of health care-associated infections. the information presented will be useful to new infection control professionals as well as persons involved in designing or responding to state programs for public reporting of hai rates. part iii describes each of the categories of precautions developed by the health care infection control practices advisory committee (hicpac) and the cdc and provides guidance for their application in various health care settings. the categories of transmission-based precautions are unchanged from those in the guideline: contact, droplet, and airborne. one important change is the recommendation to don the indicated personal protective equipment (ppe-gowns, gloves, mask) on entry into the patient's room for patients who are on contact and/or droplet precautions, because the nature of the interaction with the patient cannot be predicted with certainty, and contaminated environmental surfaces are important sources for transmission of pathogens. in addition, the pe for patients undergoing allogeneic hsct, described in previous guidelines, has been updated. five tables summarize important information. table provides a summary of the evolution of this document. table gives guidance on using empiric isolation precautions according to a clinical syndrome. table summarizes infection control recommendations for cdc category a agents of bioterrorism. table lists the components of standard precautions and recommendations for their application, and table lists components of the pe. a glossary of definitions used in this guideline also is provided. new to this edition of the guideline is a figure showing the recommended sequence for donning and removing ppe used for isolation precautions to optimize safety and prevent self-contamination during removal. appendix a provides an updated alphabetical list of most infectious agents and clinical conditions for which isolation precautions are recommended. a preamble to the appendix provides a rationale for recommending the use of or more transmission-based precautions in addition to standard precautions, based on a review of the literature and evidence demonstrating a real or potential risk for person-to-person transmission in health care settings. the type and duration of recommended precautions are presented, with additional comments concerning the use of adjunctive measures or other relevant considerations to prevent transmission of the specific agent. relevant citations are included. new to this guideline is a comprehensive review and detailed recommendations for prevention of transmission of mdros. this portion of the guideline was published electronically in october and updated in november (siegel jd, rhinehart e, jackson m, chiarello l and hicpac. management of multidrug-resistant organisms in health care settings, ; available from http://www.cdc.gov/ ncidod/dhqp/pdf/ar/mdroguideline .pdf), and is considered a part of the guideline for isolation precautions. this section provides a detailed review of the complex topic of mdro control in health care settings and is intended to provide a context for evaluation of mdro at individual health care settings. a rationale and institutional requirements for developing an effective mdro control program are summarized. although the focus of this guideline is on measures to prevent transmission of mdros in health care settings, information concerning the judicious use of antimicrobial agents also is presented, because such practices are intricately related to the size of the reservoir of mdros, which in turn influences transmission (eg, colonization pressure). two tables summarize recommended prevention and control practices using categories of interventions to control mdros: administrative measures, education of hcws, judicious antimicrobial use, surveillance, infection control precautions, environmental measures, and decolonization. recommendations for each category apply to and are adapted for the various health care settings. with the increasing incidence and prevalence of mdros, all health care facilities must prioritize effective control of mdro transmission. facilities should identify prevalent mdros at the facility, implement control measures, assess the effectiveness of control programs, and demonstrate decreasing mdro rates. a set of intensified mdro prevention interventions is to be added if the incidence of transmission of a target mdro is not decreasing despite implementation of basic mdro infection control measures, and when the first case of an epidemiologically important mdro is identified within a health care facility. this updated guideline responds to changes in health care delivery and addresses new concerns about transmission of infectious agents to patients and hcws in the united states and infection control. the primary objective of the guideline is to improve the safety of the nation's health care delivery system by reducing the rates of hais. instruct symptomatic persons to cover mouth/nose when sneezing/ coughing; use tissues and dispose in no-touch receptacle; observe hand hygiene after soiling of hands with respiratory secretions; wear surgical mask if tolerated or maintain spatial separation, . feet if possible. *during aerosol-generating procedures on patients with suspected or proven infections transmitted by respiratory aerosols (eg, severe acute respiratory syndrome), wear a fittested n or higher respirator in addition to gloves, gown, and face/eye protection. -proper construction of windows, doors, and intake and exhaust ports -ceilings: smooth, free of fissures, open joints, crevices -walls sealed above and below the ceiling -if leakage detected, locate source and make necessary repairs d ventilation to maintain $ air changes/hour d directed air flow; air supply and exhaust grills located so that clean, filtered air enters from one side of the room, flows across the patient's bed, and exits on opposite side of the room d positive room air pressure in relation to the corridor; pressure differential of . . pa ( . -inch water gauge) d air flow patterns monitored and recorded daily using visual methods (eg, flutter strips, smoke tubes) or a hand-held pressure gauge d self-closing door on all room exits d back-up ventilation equipment (eg, portable units for fans or filters) maintained for emergency provision of ventilation requirements for pe areas, with immediate steps taken to restore the fixed ventilation system d for patients who require both a pe and an airborne infection isolation room (aiir), use an anteroom to ensure proper air balance relationships and provide independent exhaust of contaminated air to the outside, or place a hepa filter in the exhaust duct. ( ) reaffirm standard precautions as the foundation for preventing transmission during patient care in all health care settings; ( ) reaffirm the importance of implementing transmission-based precautions based on the clinical presentation or syndrome and likely pathogens until the infectious etiology has been determined ( table ) ; and ( ) provide epidemiologically sound and, whenever possible, evidence-based recommendations. this guideline is designed for use by individuals who are charged with administering infection control programs in hospitals and other health care settings. the information also will be useful for other hcws, health care administrators, and anyone needing information about infection control measures to prevent transmission of infectious agents. commonly used abbreviations are provided, and terms used in the guideline are defined in the glossary. medline and pubmed were used to search for relevant studies published in english, focusing on those published since . much of the evidence cited for preventing transmission of infectious agents in health care settings is derived from studies that used ''quasiexperimental designs,'' also referred to as nonrandomized preintervention and postintervention study designs. although these types of studies can provide valuable information regarding the effectiveness of various interventions, several factors decrease the certainty of attributing improved outcome to a specific intervention. these include: difficulties in controlling for important confounding variables, the use of multiple interventions during an outbreak, and results that are explained by the statistical principle of regression to the mean (eg, improvement over time without any intervention). observational studies remain relevant and have been used to evaluate infection control interventions. , the quality of studies, consistency of results, and correlation with results from randomized controlled trials, when available, were considered during the literature review and assignment of evidencebased categories (see part iv: recommendations) to the recommendations in this guideline. several authors have summarized properties to consider when evaluating studies for the purpose of determining whether the results should change practice or in designing new studies. , , this guideline contains changes in terminology from the guideline: . the term ''nosocomial infection'' is retained to refer only to infections acquired in hospitals. the term ''health care-associated infection'' (hai) is used to refer to infections associated with health care delivery in any setting (eg, hospitals, long-term care facilities, ambulatory settings, home care). this term reflects the inability to determine with certainty where the pathogen was acquired, because patients may be colonized with or exposed to potential pathogens outside of the health care setting before receiving health care, or may develop infections caused by those pathogens when exposed to the conditions associated with delivery of health care. in addition, patients frequently move among the various settings within the health care system. of infectious agents, a susceptible host with a portal of entry receptive to the agent, and a mode of transmission for the agent. this section describes the interrelationship of these elements in the epidemiology of hais. i.b. . sources of infectious agents. infectious agents transmitted during health care derive primarily from human sources but inanimate environmental sources also are implicated in transmission. human reservoirs include patients, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] hcws, , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and household members and other visitors. [ ] [ ] [ ] [ ] [ ] [ ] such source individuals may have active infections, may be in the asymptomatic and/or incubation period of an infectious disease, or may be transiently or chronically colonized with pathogenic microorganisms, particularly in the respiratory and gastrointestinal tracts. other sources of hais are the endogenous flora of patients (eg, bacteria residing in the respiratory or gastrointestinal tract). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] i.b. . susceptible hosts. infection is the result of a complex interrelationship between a potential host and an infectious agent. most of the factors that influence infection and the occurrence and severity of disease are related to the host. however, characteristics of the host-agent interaction as it relates to pathogenicity, virulence, and antigenicity also are important, as are the infectious dose, mechanisms of disease production, and route of exposure. there is a spectrum of possible outcomes after exposure to an infectious agent. some persons exposed to pathogenic microorganisms never develop symptomatic disease, whereas others become severely ill and even die. some individuals are prone to becoming transiently or permanently colonized but remain asymptomatic. still others progress from colonization to symptomatic disease either immediately after exposure or after a period of asymptomatic colonization. the immune state at the time of exposure to an infectious agent, interaction between pathogens, and virulence factors intrinsic to the agent are important predictors of an individual's outcome. host factors such as extremes of age and underlying disease (eg, diabetes , , human immunodeficiency virus/acquired immune deficiency syndrome [hiv/ aids], , malignancy, and transplantation , , ) can increase susceptibility to infection, as can various medications that alter the normal flora (eg, antimicrobial agents, gastric acid suppressors, corticosteroids, antirejection drugs, antineoplastic agents, immunosuppressive drugs). surgical procedures and radiation therapy impair defenses of the skin and other involved organ systems. indwelling devices, such as urinary catheters, endotracheal tubes, central venous and arterial catheters, [ ] [ ] [ ] and synthetic implants, facilitate development of hais by allowing potential pathogens to bypass local defenses that ordinarily would impede their invasion and by providing surfaces for development of biofilms that may facilitate adherence of microorganisms and protect from antimicrobial activity. some infections associated with invasive procedures result from transmission within the health care facility; others arise from the patient's endogenous flora. clothing, uniforms, laboratory coats, or isolation gowns used as ppe may become contaminated with potential pathogens after care of a patient colonized or infected with an infectious agent, (eg, mrsa, vancomycin-resistant enterococci [vre], and c difficile ). although contaminated clothing has not been implicated directly in transmission, the potential exists for soiled garments to transfer infectious agents to successive patients. i.b. .b. droplet transmission. droplet transmission is technically a form of contact transmission; some infectious agents transmitted by the droplet route also may be transmitted by direct and indirect contact routes. however, in contrast to contact transmission, respiratory droplets carrying infectious pathogens transmit infection when they travel directly from the respiratory tract of the infectious individual to susceptible mucosal surfaces of the recipient, generally over short distances, necessitating facial protection. respiratory droplets are generated when an infected person coughs, sneezes, or talks , or during such procedures as suctioning, endotracheal intubation, [ ] [ ] [ ] [ ] cough induction by chest physiotherapy, and cardiopulmonary resuscitation. , evidence for droplet transmission comes from epidemiologic studies of disease outbreaks, [ ] [ ] [ ] [ ] from experimental studies, and from information on aerosol dynamics. , studies have shown that the nasal mucosa, conjunctivae, and, less frequently, the mouth are susceptible portals of entry for respiratory viruses. the maximum distance for droplet transmission is currently unresolved; pathogens transmitted by the droplet route have not been transmitted through the air over long distances, in contrast to the airborne pathogens discussed below. historically, the area of defined risk has been a distance of , feet around the patient, based on epidemiologic and simulated studies of selected infections. , using this distance for donning masks has been effective in preventing transmission of infectious agents through the droplet route. however, experimental studies with smallpox , and investigations during the global sars outbreaks of suggest that droplets from patients with these infections could reach persons located feet or more from their source. it is likely that the distance that droplets travel depends on the velocity and mechanism by which respiratory droplets are propelled from the source, the density of respiratory secretions, environmental factors (eg, temperature, humidity), and the pathogen's ability to maintain infectivity over that distance. thus, a distance of , feet around the patient is best considered an example of what is meant by ''a short distance from a patient'' and should not be used as the sole criterion for determining when a mask should be donned to protect from droplet exposure. based on these considerations, it may be prudent to don a mask when within to feet of the patient or on entry into the patient's room, especially when exposure to emerging or highly virulent pathogens is likely. more studies are needed to gain more insight into droplet transmission under various circumstances. droplet size is another variable under investigation. droplets traditionally have been defined as being . mm in size. droplet nuclei (ie, particles arising from desiccation of suspended droplets) have been associated with airborne transmission and defined as , mm in size, a reflection of the pathogenesis of pulmonary tuberculosis that is not generalizeable to other organisms. observations of particle dynamics have demonstrated that a range of droplet sizes, including those of diameter $ mm, can remain suspended in the air. the behavior of droplets and droplet nuclei affect recommendations for preventing transmission. whereas fine airborne particles containing pathogens that are able to remain infective may transmit infections over long distances, requiring aiir to prevent its dissemination within a facility; organisms transmitted by the droplet route do not remain infective over long distances and thus do not require special air handling and ventilation. examples of infectious agents transmitted through the droplet route include b pertussis, influenza virus, adenovirus, rhinovirus, mycoplasma pneumoniae, sars-cov, , , group a streptococcus, and neisseria meningitides. , , although rsv may be transmitted by the droplet route, direct contact with infected respiratory secretions is the most important determinant of transmission and consistent adherence to standard precautions plus contact precautions prevents transmission in health care settings. , , rarely, pathogens that are not transmitted routinely by the droplet route are dispersed into the air over short distances. for example, although s aureus is transmitted most frequently by the contact route, viral upper respiratory tract infection has been associated with increased dispersal of s aureus from the nose into the air for a distance of feet under both outbreak and experimental conditions; this is known as the ''cloud baby'' and ''cloud adult'' phenomenon. [ ] [ ] [ ] i.b. .c. airborne transmission. airborne transmission occurs by dissemination of either airborne droplet nuclei or small particles in the respirable size range containing infectious agents that remain infective over time and distance (eg, spores of aspergillus spp and m tuberculosis). microorganisms carried in this manner may be dispersed over long distances by air currents and may be inhaled by susceptible individuals who have not had face-to-face contact with (or even been in the same room with) the infectious individual. [ ] [ ] [ ] [ ] preventing the spread of pathogens that are transmitted by the airborne route requires the use of special air handling and ventilation systems (eg, aiirs) to contain and then safely remove the infectious agent. , infectious agents to which this applies include m tuberculosis, - rubeola virus (measles), and varicella-zoster virus (chickenpox). in addition, published data suggest the possibility that variola virus (smallpox) may be transmitted over long distances through the air under unusual circumstances, and aiirs are recommended for this agent as well; however, droplet and contact routes are the more frequent routes of transmission for smallpox. , , in addition to aiirs, respiratory protection with a national institute for occupational safety and health (niosh)-certified n or higher-level respirator is recommended for hcws entering the aiir, to prevent acquisition of airborne infectious agents such as m tuberculosis. for certain other respiratory infectious agents, such as influenza , and rhinovirus, and even some gastrointestinal viruses (eg, norovirus and rotavirus ) , there is some evidence that the pathogen may be transmitted through small-particle aerosols under natural and experimental conditions. such transmission has occurred over distances . feet but within a defined air space (eg, patient room), suggesting that it is unlikely that these agents remain viable on air currents that travel long distances. aiirs are not routinely required to prevent transmission of these agents. additional issues concerning small-particle aerosol transmission of agents that are most frequently transmitted by the droplet route are discussed below. although sars-cov is transmitted primarily by contact and/or droplet routes, airborne transmission over a limited distance (eg, within a room) has been suggested, although not proven. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] this is true of other infectious agents as well, such as influenza virus and noroviruses. , , influenza viruses are transmitted primarily by close contact with respiratory droplets, , and acquisition by hcws has been prevented by droplet precautions, even when positive-pressure rooms were used in one center. however, inhalational transmission could not be excluded in an outbreak of influenza in the passengers and crew of an aircraft. observations of a protective effect of ultraviolet light in preventing influenza among patients with tuberculosis during the influenza pandemic of - have been used to suggest airborne transmission. , in contrast to the strict interpretation of an airborne route for transmission (ie, long distances beyond the patient room environment), short-distance transmission by small-particle aerosols generated under specific circumstances (eg, during endotracheal intubation) to persons in the immediate area near the patient also has been demonstrated. aerosolized particles , mm in diameter can remain suspended in air when room air current velocities exceed the terminal settling velocities of the particles. sars-cov transmission has been associated with endotracheal intubation, noninvasive positive pressure ventilation, and cardiopulmonary resuscitation. , , , , although the most frequent routes of transmission of noroviruses are contact and foodborne and waterborne routes, several reports suggest that noroviruses also may be transmitted through aerosolization of infectious particles from vomitus or fecal material. , , , it is hypothesized that the aerosolized particles are inhaled and subsequently swallowed. roy this conceptual framework can explain rare occurrences of airborne transmission of agents that are transmitted most frequently by other routes (eg, smallpox, sars, influenza, noroviruses). concerns about unknown or possible routes of transmission of agents associated with severe disease and no known treatment often result in the adoption of overextreme prevention strategies, and recommended precautions may change as the epidemiology of an emerging infection becomes more well defined and controversial issues are resolved. i.b. .d.ii. transmission from the environment. some airborne infectious agents are derived from the environment and do not usually involve person-to-person transmission; for example, anthrax spores present in a finely milled powdered preparation can be aerosolized from contaminated environmental surfaces and inhaled into the respiratory tract. , spores of environmental fungi (eg, aspergillus spp) are ubiquitous in the environment and may cause disease in immunocompromised patients who inhale aerosolized spores (through, eg, construction dust). , as a rule, neither of these organisms is subsequently transmitted from infected patients; however, there is well-documented report of person-to-person transmission of aspergillus sp in the icu setting that was most likely due to the aerosolization of spores during wound debridement. the pe involves isolation practices designed to decrease the risk of exposure to environmental fungal agents in allogeneic hsct patients. , , , [ ] [ ] [ ] [ ] environmental sources of respiratory pathogens (eg, legionella) transmitted to humans through a common aerosol source is distinct from direct patient-to-patient transmission. i.b. .e. other sources of infection. sources of infection transmission other than infectious individuals include those associated with common environmental sources or vehicles (eg, contaminated food, water, or medications, such as intravenous fluids). although aspergillus spp have been recovered from hospital water systems, the role of water as a reservoir for immunosuppressed patients remains unclear. vectorborne transmission of infectious agents from mosquitoes, flies, rats, and other vermin also can occur in health care settings. prevention of vectorborne transmission is not addressed in this document. this section discusses several infectious agents with important infection control implications that either were not discussed extensively in previous isolation s vol. no. supplement guidelines or have emerged only recently. included are epidemiologically important organisms (eg, c difficile), agents of bioterrorism, prions, sars-cov, monkeypox, noroviruses, and the hemorrhagic fever viruses (hfvs). experience with these agents has broadened the understanding of modes of transmission and effective preventive measures. these agents are included for information purposes and, for some (ie, sars-cov, monkeypox), to highlight the lessons that have been learned about preparedness planning and responding effectively to new infectious agents. i.c. . epidemiologically important organisms. under defined conditions, any infectious agent transmitted in a health care setting may become targeted for control because it is epidemiologically important. c difficile is specifically discussed below because of its current prevalence and seriousness in us health care facilities. in determining what constitutes an ''epidemiologically important organism,'' the following criteria apply: d a propensity for transmission within health care facilities based on published reports and the occurrence of temporal or geographic clusters of more than patients, (eg, c difficile, norovirus, rsv, influenza, rotavirus, enterobacter spp, serratia spp, group a streptococcus). a single case of health care-associated invasive disease caused by certain pathogens (eg, group a streptococcus postoperatively, in a burn unit, or in a ltcf; legionella spp, , aspergillus spp ) is generally considered a trigger for investigation and enhanced control measures because of the risk of additional cases and the severity of illness associated with these infections. i.c. .a. clostridium difficile. c difficile is a sporeforming gram-positive anaerobic bacillus that was first isolated from stools of neonates in and identified as the most frequent causative agent of antibioticassociated diarrhea and pseudomembranous colitis in . this pathogen is a major cause of health care-associated diarrhea and has been responsible for many large outbreaks in health care settings that have proven extremely difficult to control. important factors contributing to health care-associated outbreaks include environmental contamination, persistence of spores for prolonged periods, resistance of spores to routinely used disinfectants and antiseptics, hand carriage by hcws to other patients, and exposure of patients to frequent courses of antimicrobial agents. antimicrobials most frequently associated with increased risk of c difficile include third-generation cephalosporins, clindamycin, vancomycin, and fluoroquinolones. since , outbreaks and sporadic cases of c difficile with increased morbidity and mortality have occurred in several us states, canada, england, and the netherlands. [ ] [ ] [ ] [ ] [ ] the same strain of c difficile has been implicated in all of these outbreaks; this strain, toxinotype iii, north american pulsedfield gel electrophoresis (pfge) type , and polymerase chain reaction (pcr)-ribotype (nap / ), has been found to hyperproduce toxin a (a -fold increase) and toxin b (a -fold increase) compared with isolates from other pfge types. a recent survey of us infectious disease physicians found that % of the respondents perceived recent increases in the incidence and severity of c difficile disease. standardization of testing methodology and surveillance definitions is needed for accurate comparisons of trends in rates among hospitals. it is hypothesized that the incidence of disease and apparent heightened transmissibility of this new strain may be due, at least in part, to the greater production of toxins a and b, increasing the severity of diarrhea and producing more environmental contamination. considering the greater morbidity, mortality, length of stay, and costs associated with c difficile disease in both acute care and long-term care facilities, control of this pathogen is becoming increasingly important. prevention of transmission focuses on syndromic application of contact precautions for patients with diarrhea, accurate identification of affected patients, environmental measures (eg, rigorous cleaning of patient rooms), and consistent hand hygiene. using soap and water rather than alcohol-based handrubs for mechanical removal of spores from hands and using a bleachcontaining disinfectant ( ppm) for environmental disinfection may be valuable in cases of transmission in health care facilities. appendix a provides for recommendations. i.c. .b. multidrug-resistant organisms. in general, mdros are defined as microorganisms-predominantly bacteria-that are resistant to or more classes of antimicrobial agents. although the names of certain mdros suggest resistance to only a single agent (eg, mrsa, vre), these pathogens are usually resistant to all but a few commercially available antimicrobial agents. this latter feature defines mdros that are considered to be epidemiologically important and deserve special attention in health care facilities. other mdros of current concern include multidrug-resistant streptococcus pneumoniae, which is resistant to penicillin and other broad-spectrum agents such as macrolides and fluroquinolones, multidrug-resistant gram-negative bacilli (mdr-gnb), especially those producing esbls; and strains of s aureus that are intermediate or resistant to vancomycin (ie, visa and vrsa). mdros are transmitted by the same routes as antimicrobial susceptible infectious agents. patient-to-patient transmission in health care settings, usually via hands of hcws, has been a major factor accounting for the increase in mdro incidence and prevalence, especially for mrsa and vre in acute care facilities. [ ] [ ] [ ] preventing the emergence and transmission of these pathogens requires a comprehensive approach that includes administrative involvement and measures (eg, nurse staffing, communication systems, performance improvement processes to ensure adherence to recommended infection control measures), education and training of medical and other hcws, judicious antibiotic use, comprehensive surveillance for targeted mdros, application of infection control precautions during patient care, environmental measures (eg, cleaning and disinfection of the patient care environment and equipment, dedicated single-patient use of noncritical equipment), and decolonization therapy when appropriate. the prevention and control of mdros is a national priority, one that requires that all health care facilities and agencies assume responsibility and participate in community-wide control programs. , a detailed discussion of this topic and recommendations for prevention published in is available at http:// www.cdc.gov/ncidod/dhqp/pdf/ar/mdroguideline . pdf. i.c. . agents of bioterrorism. the cdc has designated the agents that cause anthrax, smallpox, plague, tularemia, viral hemorrhagic fevers, and botulism as category a (high priority), because these agents can be easily disseminated environmentally and/or transmitted from person to person, can cause high mortality and have the potential for major public health impact, might cause public panic and social disruption, and necessitate special action for public health preparedness. general information relevant to infection control in health care settings for category a agents of bioterrorism is summarized in table . (see http:// www.bt.cdc.gov for additional, updated category a agent information as well as information concerning category b and c agents of bioterrorism and updates.) category b and c agents are important but are not as readily disseminated and cause less morbidity and mortality than category a agents. health care facilities confront a different set of issues when dealing with a suspected bioterrorism event compared with other communicable diseases. an understanding of the epidemiology, modes of transmission, and clinical course of each disease, as well as carefully drafted plans that specify an approach and relevant websites and other resources for disease-specific guidance to health care, administrative, and support personnel, are essential for responding to and managing a bioterrorism event. infection control issues to be addressed include ( ) identifying persons who may be exposed or infected; ( ) preventing transmission among patients, hcws, and visitors; ( ) providing treatment, chemoprophylaxis, or vaccine to potentially large numbers of people; ( ) protecting the environment, including the logistical aspects of securing sufficient numbers of aiirs or designating areas for patient cohorts when an insufficient number of aiirs is available; ( ) providing adequate quantities of appropriate ppe; and ( ) identifying appropriate staff to care for potentially infectious patients (eg, vaccinated hcws for care of patients with smallpox). the response is likely to differ for exposures resulting from an intentional release compared with a naturally occurring disease because of the large number of persons that can be exposed at the same time and possible differences in pathogenicity. various sources offer guidance for the management of persons exposed to the most likely agents of bioterrorism. federal agency websites (eg, http://www. usamriid.army.mil/publications/index.html and http:// www.bt.cdc.gov) and state and county health department websites should be consulted for the most upto-date information. sources of information on specific agents include anthrax, smallpox, [ ] [ ] [ ] plague, , botulinum toxin, tularemia, and hemorrhagic fever viruses. , i.c. .a. pre-event administration of smallpox (vaccinia) vaccine to health care workers. vaccination of hcwsl in preparation for a possible smallpox exposure has important infection control implications. [ ] [ ] [ ] these include the need for meticulous screening for vaccine contraindications in persons at increased risk for adverse vaccinia events; containment and monitoring of the vaccination site to prevent transmission in the health care setting and at home; and management of patients with vaccinia-related adverse events. , the pre-event us smallpox vaccination program of is an example of the effectiveness of carefully developed recommendations for both screening potential vaccinees for contraindications and vaccination site care and monitoring. between december and february , approximately , individuals were vaccinated in the department of defense and , in the civilian or public health populations, including approximately , who worked in health care settings. no cases of eczema vaccinatum, progressive vaccinia, fetal vaccinia, or contact transfer of vaccinia were reported in health care settings or in military workplaces. , outside the health care setting, there were cases of contact transfer from military vaccinees to close personal contacts (eg, bed partners or contacts during participation in sports such as wrestling ). all contact transfers were from individuals who were not following recommendations to cover their vaccination sites. vaccinia virus was confirmed by culture or pcr in cases, of which resulted from tertiary transfer. all recipients, including breast-fed infant, recovered without complications. subsequent studies using viral culture and pcr techniques have confirmed the effectiveness of semipermeable dressings to contain vaccinia. [ ] [ ] [ ] [ ] this experience emphasizes the importance of ensuring that newly vaccinated hcws adhere to recommended vaccination site care, especially those caring for high-risk patients. recommendations for pre-event smallpox vaccination of hcws and vacciniarelated infection control recommendations are published in the morbidity and mortality weekly report, , with updates posted on the cdc's bioterrorism website. i.c. . prions. creutzfeldt-jakob disease (cjd) is a rapidly progressive, degenerative neurologic disorder of humans, with an incidence in the united states of approximately person/million population/year. , cjd is believed to be caused by a transmissible proteinaceous infectious agent known as a prion. infectious prions are isoforms of a host-encoded glycoprotein known as the prion protein. the incubation period (ie, time between exposure and and onset of symptoms) varies from years to many decades. however, death typically occurs within year of the onset of symptoms. approximately % of cjd cases occur sporadically with no known environmental source of infection, and % of cases are familial. iatrogenic transmission has occurred, with most cases resulting from treatment with human cadaver pituitary-derived growth hormone or gonadotropin, , from implantation of contaminated human dura mater grafts, or from corneal transplants. transmission has been linked to the use of contaminated neurosurgical instruments or stereotactic electroencephalogram electrodes. [ ] [ ] [ ] [ ] prion diseases in animals include scrapie in sheep and goats, bovine spongiform encephalopathy (bse, or ''mad cow disease'') in cattle, and chronic wasting disease in deer and elk. bse, first recognized in the united kingdom in , was associated with a major epidemic among cattle that had consumed contaminated meat and bone meal. the possible transmission of bse to humans causing variant cjd (vcjd) was first described in and was subsequently found to be associated with consumption of bse-contaminated cattle products primarily in the united kingdom. there is strong epidemiologic and laboratory evidence for a causal association between the causative agent of bse and vcjd. although most cases of vcjd have been reported from the united kingdom, a few cases also have been reported from europe, japan, canada, and the united states. most persons affected with vcjd worldwide lived in or visited the united kingdom during the years of a large outbreak of bse ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and may have consumed contaminated cattle products during that time (see http://www.cdc.gov/ncidod/ diseases/cjd/cjd.htm). although there has been no indigenously acquired vcjd in the united states, the sporadic occurrence of bse in cattle in north america has heightened awareness of the possibility that such infections could occur and have led to increased surveillance activities. updated information may be found at http://www.cdc.gov/ncidod/diseases/cjd/cjd.htm. the public health impact of prion diseases has been reviewed previously. vcjd in humans has different clinical and pathologic characteristics than sporadic or classic cjd, including ( ) younger median age at death ( [range, to ] vs years), ( ) longer median duration of illness ( months vs to months), ( ) increased frequency of sensory symptoms and early psychiatric symptoms with delayed onset of frank neurologic signs; and ( ) detection of prions in tonsillar and other lymphoid tissues, not present in sporadic cjd. similar to sporadic cjd, there have been no reported cases of direct human-tohuman transmission of vcjd by casual or environmental contact, droplet, or airborne routes. ongoing blood safety surveillance in the united states has not detected sporadic cjd transmission through blood transfusion; - however, bloodborne transmission of vcjd is believed to have occurred in patients in the uited kingdom. , the following fda websites provide information on steps currently being taken in the united states to protect the blood supply from cjd and vcjd: http://www.fda.gov/cber/gdlns/cjdvcjd.htm and http:// www.fda.gov/cber/gdlns/cjdvcjdq&a.htm. standard precautions are used when caring for patients with suspected or confirmed cjd or vcjd. however, special precautions are recommended for tissue handling in the histology laboratory and for conducting an autopsy, embalming, and coming into contact with a body that has undergone autopsy. recommendations for reprocessing surgical instruments to prevent transmission of cjd in health care settings have been published by the world health organization (who) and are currently under review at the cdc. questions may arise concerning notification of patients potentially exposed to cjd or vcjd through contaminated instruments and blood products from patients with cjd or vcjd or at risk of having vcjd. the risk of transmission associated with such exposures is believed to be extremely low but may vary based on the specific circumstance. therefore, consultation on appropriate options is advised. the united kingdom has developed several documents that clinicians and patients in the united states may find useful (see http://www.hpa.org.uk/infections/topics_az/cjd/ information_documents.htm). i.c. . severe acute respiratory syndrome. sars is a newly discovered respiratory disease that emerged in china late in and spread to several countries. , in particular, mainland china, hong kong, hanoi, singapore, and toronto have been significantly affected. sars is caused by sars-cov, a previously unrecognized member of the coronavirus family. , the incubation period from exposure to the onset of symptoms is typically to days, but can be as long as days and in rare cases even longer. the illness is initially difficult to distinguish from other common respiratory infections. signs and symptoms usually include fever above . c and chills and rigors, sometimes accompanied by headache, myalgia, and mild to severe respiratory symptoms. a radiographic profile of atypical pneumonia is an important clinical indicator of possible sars. compared with adults, children are affected less frequently, have milder disease, and are less likely to transmit sars-cov. , [ ] [ ] [ ] the overall case fatality rate is approximately %; underlying disease and advanced age increase the risk of mortality (see http://www.who.int/csr/sarsarchive/ _ _ a/en/). outbreaks in health care settings, with transmission to large numbers of hcws and patients, haa been a striking feature of sars; undiagnosed infectious patients and visitors have been important initiators of these outbreaks. , [ ] [ ] [ ] the relative contribution of potential modes of transmission is not known precisely. there is ample evidence for droplet and contact transmission; , , however, opportunistic airborne transmission cannot be excluded. , [ ] [ ] [ ] [ ] [ ] , for example, exposure to aerosol-generating procedures (eg, endotracheal intubation, suctioning) has been associated with transmission of infection to large numbers of hcws outside of the united states. , , , , therefore, aerosolization of small infectious particles generated during these and other similar procedures could be a risk factor for transmission to others within a multibed room or shared airspace. a review of the infection control literature generated from the sars outbreaks of concluded that the greatest risk of transmission is to those who have close contact, are not properly trained in use of protective infection control procedures, and do not consistently use ppe, and that n or higher-level respirators may offer additional protection to those exposed to aerosol-generating procedures and high-risk activities. , organizational and individual factors that affect adherence to infection control practices for sars also were identified. control of sars requires a coordinated, dynamic response by multiple disciplines in a health care setting. early detection of cases is accomplished by screening persons with symptoms of a respiratory infection for history of travel to areas experiencing community transmission or contact with sars patients, followed by implementation of respiratory hygiene/cough etiquette (ie, placing a mask over the patient's nose and mouth) and physical separation from other patients in common waiting areas. the precise combination of precautions to protect hcws has not yet been determined. at the time of this publication, the cdc recommends standard precautions, with emphasis on the use of hand hygiene; contact precautions, with emphasis on environmental cleaning due to the detection of sars-cov rna by pcr on surfaces in rooms occupied by sars patients; , , and airborne precautions, including use of fit-tested niosh-approved n or higher-level respirators and eye protection. in hong kong, the use of droplet and contact precautions, including the use of a mask but not a respirator, was effective in protecting hcws. however, in toronto, consistent use of an n respirator was found to be slightly more protective than a mask. it is noteworthy that no transmission of sars-cov to public hospital workers occurred in vietnam despite inconsistent use of infection control measures, including use of ppe, which suggests other factors (eg, severity of disease, frequency of high-risk procedures or events, environmental features) may influence opportunities for transmission. sars-cov also has been transmitted in the laboratory setting through breaches in recommended laboratory practices. research laboratories in which sars-cov was under investigation were the source of most cases reported after the first series of outbreaks in the winter and spring of . lessons learned from the sars outbreaks are useful in devising plans to respond to future public health crises, such as pandemic influenza and bioterrorism events. surveillance for cases among patients and hcws, ensuring availability of adequate supplies and staffing, and limiting access to health care facilities were important factors in the response to sars. guidance for infection control precautions in various settings is available at http://www.cdc.gov/ncidod/sars. i.c. . monkeypox. monkeypox is a rare viral disease found mostly in the rain forest countries of central and west africa. the disease is caused by an orthopoxvirus that is similar in appearance to smallpox but causes a milder disease. the only recognized outbreak of human monkeypox in the united states was detected in june , after several people became ill after contact with sick pet prairie dogs. infection in the prairie dogs was subsequently traced to their contact with a shipment of animals from africa, including giant gambian rats. this outbreak demonstrates the importance of recognition and prompt reporting of unusual disease presentations by clinicians to enable prompt identification of the etiology, as well as the potential of epizootic diseases to spread from animal reservoirs to humans through personal and occupational exposure. only limited data on transmission of monkeypox are available. transmission from infected animals and humans is believed to occur primarily through direct contact with lesions and respiratory secretions; airborne transmission from animals to humans is unlikely but cannot be excluded, and may have occurred in veterinary practices (eg, during administration of nebulized medications to ill prairie dogs ). in humans, instances of monkeypox transmission in hospitals have been reported in africa among children, usually related to sharing the same ward or bed. , additional recent literature documents transmission of congo basin monkeypox in a hospital compound for an extended number of generations. there has been no evidence of airborne or any other person-to-person transmission of monkeypox in the united states, and no new cases of monkeypox have been identified since the outbreak in june . the outbreak strain is a clade of monkeypox distinct from the congo basin clade and may have different epidemiologic properties (including human-to-human transmission potential) from monkeypox strains of the congo basin; this awaits further study. smallpox vaccine is % protective against congo basin monkeypox. because there is an associated case fatality rate of , %, administration of smallpox vaccine within days to individuals who have had direct exposure to patients or animals with monkeypox is a reasonable policy. for the most current information on monkeypox, see http://www.cdc.gov/ncidod/mon keypox/clinicians.htm. i.c. . noroviruses. noroviruses, formerly referred to as norwalk-like viruses, are members of the caliciviridae family. these agents are transmitted via contaminated food or water and from person to person, causing explosive outbreaks of gastrointestinal disease. environmental contamination also has been documented as a contributing factor in ongoing transmission during outbreaks. , although noroviruses cannot be propagated in cell culture, dna detection by molecular diagnostic techniques has brought a greater appreciation of their role in outbreaks of gastrointestinal disease. reported outbreaks in hospitals, and large crowded shelters established for hurricane evacuees has demonstrated their highly contagious nature, their potentially disruptive impact in health care facilities and the community, and the difficulty of controlling outbreaks in settings in which people share common facilites and space. of note, there is nearly a -fold increase in the risk to patients in outbreaks when a patient is the index case compared with exposure of patients during outbreaks when a staff member is the index case. the average incubation period for gastroenteritis caused by noroviruses is to hours, and the clinical course lasts to hours. illness is characterized by acute onset of nausea, vomiting, abdominal cramps, and/or diarrhea. the disease is largely self-limited; rarely, death due to severe dehydration can occur, particularly in elderly persons with debilitating health conditions. the epidemiology of norovirus outbreaks shows that even though primary cases may result from exposure to a fecally contaminated food or water, secondary and tertiary cases often result from person-to-person transmission facilitated by contamination of fomites , and dissemination of infectious particles, especially during the process of vomiting. , , , , , , , widespread, persistent, and inapparent contamination of the environment and fomites can make outbreaks extremely difficult to control. , , these clinical observations and the detection of norovirus dna on horizontal surfaces feet above the level that might be touched normally suggest that under certain circumstances, aerosolized particles may travel distances beyond feet. it is hypothesized that infectious particles may be aerosolized from vomitus, inhaled, and swallowed. in addition, individuals who are responsible for cleaning the environment may be at increased risk of infection. development of disease and transmission may be facilitated by the low infectious dose (ie, , viral particles) and the resistance of these viruses to the usual cleaning and disinfection agents (ie, they may survive , ppm chlorine). [ ] [ ] [ ] an alternate phenolic agent that was shown to be effective against feline calicivirus was used for environmental cleaning in one outbreak. , there are insufficient data to determine the efficacy of alcohol-based hand rubs against noroviruses when the hands are not visibly soiled. absence of disease in certain individuals during an outbreak may be explained by protection from infection conferred by the b histo-blood group antigen. consultation on outbreaks of gastroenteritis is available through the cdc's division of viral and rickettsial diseases. i.c. . hemorrhagic fever viruses. hfv is a mixed group of viruses that cause serious disease with high fever, skin rash, bleeding diathesis, and, in some cases, high mortality; the resulting disease is referred to as viral hemorrhagic fever (vhf). among the more commonly known hfvs are ebola and marburg viruses (filoviridae), lassa virus (arenaviridae), crimean-congo hemorrhagic fever and rift valley fever virus (bunyaviridae), and dengue and yellow fever viruses (flaviviridae). , these viruses are transmitted to humans through contact with infected animals or via arthropod vectors. although none of these viruses is endemic in the united states, outbreaks in affected countries provide potential opportunities for importation by infected humans and animals. furthermore, there is a concern that some of these agents could be used as bioweapons. person-to-person transmission has been documented for ebola, marburg, lassa, and crimean-congo hfvs. in resource-limited health care settings, transmission of these agents to hcws, patients, and visitors has been described and in some outbreaks has accounted for a large proportion of cases. [ ] [ ] [ ] transmission within households also has been documented in individuals who had direct contact with ill persons or their body fluids, but not in those who did not have such contact. evidence concerning the transmission of hfvs has been summarized previously. , person-to-person transmission is associated primarily with direct blood and body fluid contact. percutaneous exposure to contaminated blood carries a particularly high risk for transmission and increased mortality. , the finding of large numbers of ebola viral particles in the skin and the lumina of sweat glands has raised concerns that transmission could occur from direct contact with intact skin, although epidemiologic evidence to support this is lacking. postmortem handling of infected bodies is an important risk for transmission. , , in rare situations, cases in which the mode of transmission was unexplained among individuals with no known direct contact have led to speculation that airborne transmission could have occurred. however, airborne transmission of naturally occurring hfvs in humans has not been documented. a study of airplane passengers exposed to an in-flight index case of lassa fever found no transmission to any passengers. in the laboratory setting, animals have been infected experimentally with marburg or ebola virus through direct inoculation of the nose, mouth, and/or conjunctiva , and by using mechanically generated viruscontaining aerosols. , transmission of ebola virus among laboratory primates in an animal facility has been described. the secondarily infected animals were in individual cages separated by approximately meters. although the possibility of airborne transmission was suggested, the investigators were not able to exclude droplet or indirect contact transmission in this incidental observation. guidance on infection control precautions for hvfs transmitted person-to-person have been published by the cdc , and by the johns hopkins center for civilian biodefense strategies. the most recent recommendations at the time of publication of this document were posted on the cdc website on may , . inconsistencies among the various recommendations have raised questions about the appropriate precautions to use in us hospitals. in less developed countries, outbreaks of hfvs have been controlled with basic hygiene, barrier precautions, safe injection practices, and safe burial practices. , the preponderance of evidence on hfv transmission indicates that standard, contact, and droplet precautions with eye protection are effective in protecting hcws and visitors coming in contact with an infected patient. single gloves are adequate for routine patient care; doublegloving is advised during invasive procedures (eg, surgery) that pose an increased risk of blood exposure. routine eye protection (ie goggles or face shield) is particularly important. fluid-resistant gowns should be worn for all patient contact. airborne precautions are not required for routine patient care; however, use of aiirs is prudent when procedures that could generate infectious aerosols are performed (eg, endotracheal intubation, bronchoscopy, suctioning, autopsy procedures involving oscillating saws). n or higher-level respirators may provide added protection for individuals in a room during aerosol-generating procedures ( table , appendix a). when a patient with a syndrome consistent with hemorrhagic fever also has a history of travel to an endemic area, precautions are initiated on presentation and then modified as more information is obtained ( table ) . patients with hemorrhagic fever syndrome in the setting of a suspected bioweapons attack should be managed using airborne precautions, including aiirs, because the epidemiology of a potentially weaponized hemorrhagic fever virus is unpredictable. numerous factors influence differences in transmission risks among the various health care settings. these factors include the population characteristics (eg, increased susceptibility to infections, type and prevalence of indwelling devices), intensity of care, exposure to environmental sources, length of stay, and frequency of interaction between patients/residents with each other and with hcws. these factors, as well as organizational priorities, goals, and resources, influence how different health care settings adapt transmission prevention guidelines to meet their specific needs. , infection control management decisions are informed by data regarding institutional experience/epidemiology; trends in community and institutional hais; local, regional, and national epidemiology; and emerging infectious disease threats. i.d. . hospitals. infection transmission risks are present in all hospital settings. however, certain hospital settings and patient populations have unique conditions that predispose patients to infection and merit special mention. these are often sentinel sites for the emergence of new transmission risks that may be unique to that setting or present opportunities for transmission to other settings in the hospital. i.d. .a. intensive care units. intensive care units (icus) serve patients who are immunocompromised by disease state and/or by treatment modalities, as well as patients with major trauma, respiratory failure, and other life-threatening conditions (eg, myocardial infarction, congestive heart failure, overdose, stroke, gastrointestinal bleeding, renal failure, hepatic failure, multiorgan system failure, and extremes of age). although icus account for a relatively small proportion of hospitalized patients, infections acquired in these units account for . % of all hais. in the national nosocomial infection surveillance (nnis) system, . % of hais were reported from icu and high-risk nursery (neonatal icu [nicu]) patients in (nnis, unpublished data). this patient population has increased susceptibility to colonization and infection, especially with mdros and candida spp, , because of underlying diseases and conditions, the invasive medical devices and technology used in their care (eg central venous catheters and other intravascular devices, mechanical ventilators, extracorporeal membrane oxygenation, hemodialysis/filtration, pacemakers, implantable left-ventricular assist devices), the frequency of contact with hcws, prolonged lengths of stay, and prolonged exposure to antimicrobial agents. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] furthermore, adverse patient outcomes in this setting are more severe and are associated with a higher mortality. outbreaks associated with various bacterial, fungal, and viral pathogens due to common-source and person-to-person transmissions are frequent in adult icus and pediatric icus (picus). , [ ] [ ] [ ] [ ] [ ] [ ] i.d. .b. burn units. burn wounds can provide optimal conditions for colonization, infection, and transmission of pathogens; infection acquired by burn patients is a frequent cause of morbidity and mortality. , , the risk of invasive burn wound infection is particularly high in patients with a burn injury involving . % of the total body surface area (tbsa). , infections occurring in patients with burn injuries involving , % of the tbsa are usually associated with the use of invasive devices. mssa, mrsa, enterococci (including vre), gram-negative bacteria, and candida spp are prevalent pathogens in burn infections, , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and outbreaks of these organisms have been reported. [ ] [ ] [ ] [ ] shifts over time in the predominance of pathogens causing infections in burn patients often lead to changes in burn care practices. , [ ] [ ] [ ] [ ] burn wound infections caused by aspergillus spp or other environmental molds may result from exposure to supplies contaminated during construction or to dust generated during construction or other environmental disruption. hydrotherapy equipment is an important environmental reservoir of gram-negative organisms. its use in burn care is discouraged based on demonstrated associations between the use of contaminated hydrotherapy equipment and infections. burn wound infections and colonization, as well as bloodstream infections, caused by multidrug-resistant p aeruginosa, acinetobacter baumannii, and mrsa have been associated with hydrotherapy; thus, excision of burn wounds in operating rooms is the preferred approach. advances in burn care (specifically, early excision and grafting of the burn wound, use of topical antimicrobial agents, and institution of early enteral feeding) have led to decreased infectious complications. other advances have included prophylactic antimicrobial use, selective digestive decontamination, and use of antimicrobial-coated catheters; however, few epidemiologic studies and no efficacy studies have been performed to investigate the relative benefit of these measures. there is no consensus on the most effective infection control practices to prevent transmission of infections to and from patients with serious burns (eg, single-bed rooms, laminar flow, and high-efficiency particulate air [hepa] filtration, or maintaining burn patients in a separate unit with no exposure to patients or equipment from other units ). there also is controversy regarding the need for and type of barrier precautions in the routine care of burn patients. one retrospective study demonstrated the efficacy and cost-effectiveness of a simplified barrier isolation protocol for wound colonization, emphasizing handwashing and use of gloves, caps, masks, and impermeable plastic aprons (rather than isolation gowns) for direct patient contact. however, to date no studies have determined the most effective combination of infection control precautions for use in burn settings. prospective studies in this area are needed. i.d. .c. pediatrics. studies of the epidemiology of hais in children have identified unique infection control issues in this population. , , [ ] [ ] [ ] [ ] [ ] pediatric icu patients and the lowest birth weight babies in the nicu monitored in the nnis system have had high rates of central venous catheter-associated bloodstream infections. , ) . close physical contact between hcws and infants and young children (eg. cuddling, feeding, playing, changing soiled diapers, and cleaning copious uncontrolled respiratory secretions) provides abundant opportunities for transmission of infectious material. such practices and behaviors as congregation of children in play areas where toys and bodily secretions are easily shared and rooming-in of family members with pediatric patients can further increase the risk of transmission. pathogenic bacteria have been recovered from toys used by hospitalized patients; contaminated bath toys were implicated in an outbreak of multidrug-resistant p. aeruginosa on a pediatric oncology unit. in addition, several patient factors increase the likelihood that infection will result from exposure to pathogens in health care settings (eg, immaturity of the neonatal immune system, lack of previous natural infection and resulting immunity, prevalence of patients with congenital or acquired immune deficiencies, congenital anatomic anomalies, and use of life-saving invasive devices in nicus and picus). there are theoretical concerns that infection risk will increase in association with innovative practices used in the nicu for the purpose of improving developmental outcomes, such factors include cobedding and kangaroo care, which may increase opportunity for skin-to-skin exposure of multiple gestation infants to each other and to their mothers, respectively; although the risk of infection actually may be reduced among infants receiving kangaroo care. children who attend child care centers , and pediatric rehabilitation units may increase the overall burden of antimicrobial resistance by contributing to the reservoir of ca-mrsa. [ ] [ ] [ ] [ ] [ ] [ ] patients in chronic care facilities may have increased rates of colonization with resistant garm-negative bacilli and may be sources of introduction of resistant organisms to acute care settings. i.d. . nonacute health care settings. health care is provided in various settings outside of hospitals, including long-term care facilities (ltcfs) (eg nursing homes), homes for the developmentally disabled, behavioral health service settings, rehabilitation centers, and hospices. in addition, health care may be provided in non-health care settings, such as workplaces with occupational health clinics, adult day care centers, assisted-living facilities, homeless shelters, jails and prisons, school clinics, and infirmaries. each of these settings has unique circumstances and population risks that must be considered when designing and implementing an infection control program. several of the most common settings and their particular challenges are discussed below. although this guideline does not address each setting, the principles and strategies provided herein may be adapted and applied as appropriate. i.d. .a. long-term care. the designation ltcf applies to a diverse group of residential settings, ranging from institutions for the developmentally disabled to nursing homes for the elderly and pediatric chronic care facilities. [ ] [ ] [ ] nursing homes for the elderly predominate numerically and frequently represent longterm care as a group of facilities. approximately . million americans reside in the nation's , nursing homes. estimates of hai rates of . to . per resident-care days have been reported, with a range of to per resident-care days in the more rigorous studies. [ ] [ ] [ ] [ ] [ ] the infrastructure described in the department of veterans affairs' nursing home care units is a promising example for the development of a nationwide hai surveillance system for ltcfs. lctfs are different from other health care settings in that elderly patients at increased risk for infection are brought together in one setting and remain in the facility for extended periods; for most residents, it is their home. an atmosphere of community is fostered, and residents share common eating and living areas and participate in various facility-sponsored activities. , because able residents interact freely with each other, controlling infection transmission in this setting can be challenging. a residents who is colonized or infected with certain microorganisms are in some cases restricted to his or her room. however, because of the psychosocial risks associated with such restriction, balancing psychosocial needs with infection control needs is important in the ltcf setting. , , , ) and bacteria, including group a streptococcus, , b pertussis, nonsusceptible s pneumoniae, , other mdros, and c difficile ). these pathogens can lead to substantial morbidity and mortality, as well as increased medical costs; prompt detection and implementation of effective control measures are needed. risk factors for infection are prevalent among ltcf residents. , , age-related declines in immunity may affect the response to immunizations for influenza and other infectious agents and increase the susceptibility to tuberculosis. immobility, incontinence, dysphagia, underlying chronic diseases, poor functional status, and age-related skin changes increase susceptibility to urinary, respiratory, and cutaneous and soft tissue infections, whereas malnutrition can impair wound healing. [ ] [ ] [ ] [ ] [ ] medications (eg, drugs that affect level of consciousness, immune function, gastric acid secretions, and normal flora, including antimicrobial therapy) and invasive devices (eg, urinary catheters and feeding tubes) heighten the susceptibility to infection and colonization in ltcf residents. [ ] [ ] [ ] finally, limited functional status and total dependence on hcws for activities of daily living have been identified as independent risk factors for infection , , and for colonization with mrsa , and esbl-producing klebsiella pneumoniae. several position papers and review articles provide guidance on various aspects of infection control and antimicrobial resistance in ltcfs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the centers for medicare and medicaid services has established regulations for the prevention of infection in ltcfs. because residents of ltcfs are hospitalized frequently, they can transfer pathogens between ltcfs and health care facilities in which they receive care. , [ ] [ ] [ ] [ ] this also is true for pediatric long-term care populations. pediatric chronic care facilities have been associated with the importation of extendedspectrum cephalosporin-resistant, gram-negative bacilli into a picu. children from pediatric rehabilitation units may contribute to the reservoir of community-associated mrsa. , [ ] [ ] [ ] i.d. .b. ambulatory care. over the past decade, health care delivery in the united states has shifted from the acute, inpatient hospital to various ambulatory and community-based settings, including the home. ambulatory care is provided in hospital-based outpatient clinics, nonhospital-based clinics and physicians' offices, public health clinics, free-standing dialysis centers, ambulatory surgical centers, urgent care centers, and other setting. in , there were million visits to hospital outpatient clinics and more than million visits to physicians' offices; ambulatory care now accounts for most patient encounters with the health care system. adapting transmission prevention guidelines to these settings is challenging, because patients remain in common areas for prolonged periods waiting to be seen by a health care provider or awaiting admission to the hospital, examination or treatment rooms are turned around quickly with limited cleaning, and infectious patients may not be recognized immediately. furthermore, immunocompromised patients often receive chemotherapy in infusion rooms, where they stay for extended periods along with other types of patients. little data exist on the risk of hais in ambulatory care settings, with the exception of hemodialysis centers. , , transmission of infections in outpatient settings has been reviewed in studies. [ ] [ ] [ ] goodman and solomon summarized clusters of infections associated with the outpatient setting between and . overall, clusters were associated with common source transmission from contaminated solutions or equipment, were associated with person-to-person transmission from or involving hcws, and were associated with airborne or droplet transmission among patients and health care workers. transmission of bloodborne pathogens (ie, hbv, hcv, and, rarely, hiv) in outbreaks, sometimes involving hundreds of patients, continues to occur in ambulatory settings. these outbreaks often are related to common source exposures, usually a contaminated medical device, multidose vial, or intravenous solution. , [ ] [ ] [ ] [ ] [ ] in all cases, transmission has been attributed to failure to adhere to fundamental infection control principles, including safe injection practices and aseptic technique. this subject has been reviewed, and recommended infection control and safe injection practices have been summarized. airborne transmission of m tuberculosis and measles in ambulatory settings, most often emergency departments, has been reported. , , , , [ ] [ ] [ ] measles virus was transmitted in physicians' offices and other outpatient settings during an era when immunization rates were low and measles outbreaks in the community were occurring regularly. , , rubella has been transmitted in the outpatient obstetric setting; there are no published reports of varicella transmission in the outpatient setting. in the ophthalmology setting, adenovirus type epidemic keratoconjunctivitis has been transmitted through incompletely disinfected ophthalmology equipment and/or from hcws to patients, presumably by contaminated hands. , , , [ ] [ ] [ ] [ ] preventing transmission in outpatient settings necessitates screening for potentially infectious symptomatic and asymptomatic individuals, especially those at possible risk for transmitting airborne infectious agents (eg, m tuberculosis, varicella-zoster virus, rubeola [measles]), at the start of the initial patient encounter. on identification of a potentially infectious patient, implementation of prevention measures, including prompt separation of potentially infectious patients and implementation of appropriate control measures (eg, respiratory hygiene/cough etiquette and transmission-based precautions) can decrease transmission risks. , transmission of mrsa and vre in outpatient settings has not been reported, but the association of ca-mrsa in hcws working in an outpatient hiv clinic with environmental ca-mrsa contamination in that clinic suggests the possibility of transmission in that setting. patient-to-patient transmission of burkholderia spp and p aeruginosa in outpatient clinics for adults and children with cystic fibrosis has been confirmed. , i.d. .c. home care. home care in the united states is delivered by more than , provider agencies, including home health agencies, hospices, durable medical equipment providers, home infusion therapy services, and personal care and support services providers. home care is provided to patients of all ages with both acute and chronic conditions. the scope of services ranges from assistance with activities of daily living and physical and occupational therapy to the care of wounds, infusion therapy, and chronic ambulatory peritoneal dialysis. the incidence of infection in home care patients, other than that associated with infusion therapy, has not been well studied. [ ] [ ] [ ] [ ] [ ] [ ] however, data collection and calculation of infection rates have been done for central venous catheter-associated bloodstream infections in patients receiving home infusion therapy [ ] [ ] [ ] [ ] [ ] and for the risk of blood contact through percutaneous or mucosal exposures, demonstrating that surveillance can be performed in this setting. draft definitions for home care-associated infections have been developed. transmission risks during home care are presumed to be minimal. the main transmission risks to home care patients are from an infectious home care provider or contaminated equipment; a provider also can be exposed to an infectious patient during home visits. because home care involves patient care by a limited number of personnel in settings without multiple patients or shared equipment, the potential reservoir of pathogens is reduced. infections of home care providers that could pose a risk to home care patients include infections transmitted by the airborne or droplet routes (eg, chickenpox, tuberculosis, influenza), skin infestations (eg, scabies and lice), and infections transmitted by direct or indirect contact (eg, impetigo). there are no published data on indirect transmission of mdros from one home care patient to another, although this is theoretically possible if contaminated equipment is transported from an infected or colonized patient and used on another patient. of note, investigations of the first case of visa in home care and the first reported cases of vrsa , , , found no evidence of transmission of visa or vrsa to other home care recipients. home health care also may contribute to antimicrobial resistance; a review of outpatient vancomycin use found that % of recipients did not receive prescribed antibiotics according to recommended guidelines. although most home care agencies implement policies and procedures aimed at preventing transmission of organisms, the current approach is based on the adaptation of the guideline for isolation precautions in hospitals, as well as other professional guidance. , this issue has proven very challenging to the home care industry, and practice has been inconsistent and frequently not evidence-based. for example, many home health agencies continue to observe ''nursing bag technique,'' a practice that prescribes the use of barriers between the nursing bag and environmental surfaces in the home. although the home environment may not always appear clean, the use of barriers between noncritical surfaces has been questioned. , opportunites exist to conduct research in home care related to infection transmission risks. i.d. .d. other sites of health care delivery. facilities that are not primarily health care settings but in which health care is delivered include clinics in correctional facilities and shelters. both of these settings can have suboptimal features, such as crowded conditions and poor ventilation. economically disadvantaged individuals who may have chronic illnesses and health care problems related to alcoholism, injected drug use, poor nutrition, and/or inadequate shelter often receive their primary health care at such sites. infectious diseases of special concern for transmission include tuberculosis, scabies, respiratory infections (eg, n meningitides, s pneumoniae), sexually transmitted and bloodborne diseases (eg, hiv, hbv, hcv, syphilis, gonorrhea), hepatitis a virus, diarrheal agents such as norovirus, and foodborne diseases. , [ ] [ ] [ ] [ ] a high index of suspicion for tuberculosis and ca-mrsa in these populations is needed; outbreaks in these settings or among the populations they serve have been reported. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] patient encounters in these types of facilities provide an opportunity to deliver recommended immunizations and screen for m tuberculosis infection, along with diagnosing and treating acute illnesses. recommended infection control measures in these nontraditional areas designated for health care delivery are the same as for other ambulatory care settings. therefore, these settings must be equipped to observe standard precautions and, when indicated, transmission-based precautions. as new treatments emerge for complex diseases, unique infection control challenges associated with special patient populations must be addressed. i.e. . immunocompromised patients. patients who have congenital primary immune deficiencies or acquired disease (eg. treatment-induced immune deficiencies) are at increased risk for numerous types of infections while receiving health care; these patients may be located throughout the health care facility. the specific immune system defects determine the types of infections most likely to be acquired (eg, viral infections are associated with t cell defects, and fungal and bacterial infections occur in patients who are neutropenic). as a general group, immunocompromised patients can be cared for in the same environment as other patients; however, it is always advisable to minimize exposure to other patients with transmissible infections, such as influenza and other respiratory viruses. , the use of more intense chemotherapy regimens for treatment of childhood leukemia may be associated with prolonged periods of neutropenia and suppression of other components of the immune system, extending the period of infection risk and raising the concern that additional precautions may be indicated for select groups. , with the application of newer and more intense immunosuppressive therapies for various medical conditions (eg, rheumatologic disease, , inflammatory bowel disease ), immunosuppressed patients are likely to be more widely distributed throughout a health care facility rather than localized to single patient units (eg, hematologyoncology). guidelines for preventing infections in certain groups of immunocompromised patients have been published previously. , , published data provide evidence to support placing patients undergoing allogeneic hsct in a pe. , , in addition, guidelines have been developed that address the special requirements of these immunocompromised patients, including use of antimicrobial prophylaxis and engineering controls to create a pe for the prevention of infections caused by aspergillus spp and other environmental fungi. , , as more intense chemotherapy regimens associated with prolonged periods of neutropenia or graft-versus-host disease are implemented, the period of risk and duration of environmental protection may need to be prolonged beyond the traditional days. i.e. . cystic fibrosis patients. patients with cystic fibrosis (cf) require special consideration when developing infection control guidelines. compared with other patients, cf patients require additional protection to prevent transmission from contaminated respiratory therapy equipment. [ ] [ ] [ ] [ ] [ ] such infectious agents as b cepacia complex and p aeruginosa. , , , have unique clinical and prognostic significance. in cf patients, b cepacia infection has been associated with increased morbidity and mortality, [ ] [ ] [ ] whereas delayed acquisition of chronic p aeruginosa infection may be associated with an improved long-term clinical outcome. , person-to-person transmission of b cepacia complex has been demonstrated among children and adults with cf in health care settings , and from various social contacts, most notably attendance at camps for patients with cf and among siblings with cf. successful infection control measures used to prevent transmission of respiratory secretions include segregation of cf patients from each other in ambulatory and hospital settings (including use of private rooms with separate showers), environmental decontamination of surfaces and equipment contaminated with respiratory secretions, elimination of group chest physiotherapy sessions, and disbanding of cf camps. , the cystic fibrosis foundation has published a consensus document with evidence-based recommendations for infection control practices in cf patients. i.f. new therapies associated with potentially transmissible infectious agents i.f. . gene therapy. gene therapy has has been attempted using various viral vectors, including nonreplicating retroviruses, adenoviruses, adeno-associated viruses, and replication-competent strains of poxviruses. unexpected adverse events have restricted the prevalence of gene therapy protocols. the infectious hazards of gene therapy are theoretical at this time but require meticulous surveillance due to the possible occurrence of in vivo recombination and the subsequent emergence of a transmissible genetically altered pathogen. the greatest concern attends the use of replication-competent viruses, especially vaccinia. to date, no reports have described transmission of a vector virus from a gene therapy recipient to another individual, but surveillance is ongoing. recommendations for monitoring infection control issues throughout the course of gene therapy trials have been published. [ ] [ ] [ ] i.f. . infections transmitted through blood, organs, and other tissues. the potential hazard of transmitting infectious pathogens through biologic products is a small but ever-present risk, despite donor screening. reported infections transmitted by transfusion or transplantation include west nile virus infection, cytomegalovirus infection, cjd, hepatitis c, infections with clostridium spp and group a streptococcus, malaria, babesiosis, chagas disease, lymphocytic choriomeningitis, and rabies. , therefore, it is important to consider receipt of biologic products when evaluating patients for potential sources of infection. i.f. . xenotransplantation. transplantation of nonhuman cells, tissues, and organs into humans potentially exposes patients to zoonotic pathogens. transmission of known zoonotic infections (eg, trichinosis from porcine tissue) is of concern. also of concern is the possibility that transplantation of nonhuman cells, tissues, or organs may transmit previously unknown zoonotic infections (xenozoonoses) to immunosuppressed human recipients. potential infections that potentially could accompany transplantation of porcine organs have been described previously. guidelines from the us public health service address many infectious diseases and infection control issues that surround the developing field of xenotransplantation; policies and procedures that explain how standard precautions and transmission-based precautions are applied, including systems used to identify and communicate information on patients with potentially transmissible infectious agents, are essential to ensure the success of these measures. these policies and procedures may vary according to the characteristics of the organization. a key administrative measure is the provision of fiscal and human resources for maintaining infection control and occupational health programs that are responsive to emerging needs. specific components include bedside nurse and infection prevention and control professional (icp) staffing levels, inclusion of icps in facility construction and design decisions, clinical microbiology laboratory support, , adequate supplies and equipment including facility ventilation systems, adherence monitoring, assessment and correction of system failures that contribute to transmission, , and provision of feedback to hcws and senior administrators. , , , the positive influence of institutional leadership has been demonstrated repeatedly in studies of hcws' adherence to recommended hand hygiene practices. , , , , , [ ] [ ] [ ] [ ] [ ] [ ] health care administrators' involvement in the infection control processes can improve their awareness of the rationale and resource requirements for following recommended infection control practices. several administrative factors may affect the transmission of infectious agents in health care settings, including the institutional culture, individual hcw behavior, and the work environment. each of these areas is suitable for performance improvement monitoring and incorporation into the organization's patient safety goals. , , , ii.a. .a. scope of work and staffing needs for infection control professionals. the effectiveness of infection surveillance and control programs in preventing nosocomial infections in ust hospitals was assessed by the cdc through the study on the efficacy of nosocomial infection control (senic project) conducted between and . in a representative sample of us general hospitals, those with a trained infection control physician or microbiologist involved in an infection control program and at least infection control nurse per beds were associated with a % lower rate of the infections studied (cvc-associated bloodstream infections, ventilator-associated pneumonias, catheter-related urinary tract infections, and surgical site infections). since the publication of that landmark study, responsibilities of icps have expanded commensurate with the growing complexity of the health care system, the patient populations served, and the increasing numbers of medical procedures and devices used in all types of health care settings. the scope of work of icps was first assessed in - by the certification board of infection control, and has been reassessed every years since that time. , [ ] [ ] [ ] the findings of these analyses have been used to develop and update the infection control certification examination, which was first offered in . with each new survey, it becomes increasingly apparent that the role of the icp is growing in complexity and scope beyond traditional infection control activities in acute care hospitals. activities currently assigned to icps in response to emerging challenges include ( ) surveillance and infection prevention at facilities other than acute care hospitals (eg, ambulatory clinics, day surgery centers, ltcfs, rehabilitation centers, home care); ( ) oversight of employee health services related to infection prevention (eg, assessment of risk and administration of recommended treatment after exposure to infectious agents, tuberculosis screening, influenza vaccination, respiratory protection fit testing, and administration of other vaccines as indicated, such as smallpox vaccine in ); ( ) preparedness planning for annual influenza outbreaks, pandemic influenza, sars, and bioweapons attacks; ( ) adherence monitoring for selected infection control practices; ( ) oversight of risk assessment and implementation of prevention measures associated with construction and renovation; ( ) prevention of transmission of mdros; ( ) evaluation of new medical products that could be associated with increased infection risk (eg, intravenous infusion materials); ( ) communication with the public, facility staff, and state and local health departments concerning infection control-related issues; and ( ) participation in local and multicenter research projects. , , , , , none of the certification board of infection control job analyses addressed specific staffing requirements for the identified tasks, although the surveys did include information about hours worked; the survey included the number of icps assigned to the responding facilities. there is agreement in the literature that a ratio of icp per acute care beds is no longer adequate to meet current infection control needs; a delphi project that assessed staffing needs of infection control programs in the st century concluded that a ratio of . to . icp per occupied acute care beds is an appropriate staffing level. a survey of participants in the nnis system found an average daily patient census of per icp. results of other studies have been similar: per beds for large acute care hospitals, per to beds in ltcfs, and . per in small rural hospitals. , the foregoing demonstrates that infection control staffing no longer can be based on patient census alone, but rather must be determined by the scope of the program, characteristics of the patient population, complexity of the health care system, tools available to assist personnel to perform essential tasks (eg, electronic tracking and laboratory support for surveillance), and unique or urgent needs of the institution and community. furthermore, appropriate training is required to optimize the quality of work performed. , , ii.a. .a.i. infection control nurse liaison. designating a bedside nurse on a patient care unit as an infection control liaison or ''link nurse'' is reported to be an effective adjunct to enhance infection control at the unit level. [ ] [ ] [ ] [ ] [ ] [ ] such individuals receive training in basic infection control and have frequent communication with icps, but maintain their primary role as bedside caregiver on their units. the infection control nurse liaison increases the awareness of infection control at the unit level. he or she is especially effective in implementating new policies or control interventions because of the rapport with individuals on the unit, an understanding of unit-specific challenges, and ability to promote strategies that are most likely to be successful in that unit. this position is an adjunct to, not a replacement for, fully trained icps. furthermore, the infection control liaison nurses should not be counted when considering icp staffing. there is increasing evidence that the level of bedside nurse staffing influences the quality of patient care. , adequate nursing staff makes it more likely that infection control practices, including hand hygiene, standard precautions, and transmission-based precautions, will be given appropriate attention and applied correctly and consistently. a national multicenter study reported strong and consistent inverse relationships between nurse staffing and adverse outcomes in medical patients, of which were hais (urinary tract infections and pneumonia). the association of nursing staff shortages with increased rates of hai has been demonstrated in several outbreaks in hospitals and ltcfs, and with increased transmission of hepatitis c virus in dialysis units. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in most cases, when staffing was improved as part of a comprehensive control intervention, the outbreak ended or the hai rate declined. in studies, , the composition of the nursing staff (''pool'' or ''float'' vs regular staff nurses) influenced the rate of primary bloodstream infections, with an increased infection rate occurring when the proportion of regular nurses decreased and that of pool nurses increased. ii.a. .c. clinical microbiology laboratory support. the critical role of the clinical microbiology laboratory in infection control and health care epidemiology has been well described , , [ ] [ ] [ ] and is supported by the infectious disease society of america's policy statement on the consolidation of clinical microbiology laboratories published in . the clinical microbiology laboratory contributes to preventing transmission of infectious diseases in health care settings by promptly detecting and reporting epidemiologically important organisms, identifying emerging patterns of antimicrobial resistance, and assessing the effectiveness of recommended precautions to limit transmission during outbreaks. outbreaks of infections may be recognized first by laboratorians. health care organizations need to ensure the availability of the recommended scope and quality of laboratory services, a sufficient number of appropriately trained laboratory staff members, and systems to promptly communicate epidemiologically important results to those who will take action (eg, providers of clinical care, infection control staff, health care epidemiologists, and infectious disease consultants). as concerns about emerging pathogens and bioterrorism grow, the role of the clinical microbiology laboratory assumes ever-greater importance. for health care organizations that outsource microbiology laboratory services (eg, ambulatory care, home care, ltcfs, smaller acute care hospitals), it is important to specify by contract the types of services (eg, periodic institution-specific aggregate susceptibility reports) required to support infection control. several key functions of the clinical microbiology laboratory are relevant to this guideline: ii.a. . institutional safety culture and organizational characteristics. safety culture (or safety climate) refers to a work environment in which a shared commitment to safety on the part of management and the workforce is understood and maintained. , , the authors of the institute of medicine's report titled to err is human acknowledged that causes of medical error are multifaceted but emphasized the pivotal role of system failures and the benefits of a safety culture. a safety culture is created through ( ) the actions that management takes to improve patient and worker safety, ( ) worker participation in safety planning, ( ) the availability of appropriate ppe, ( ) the influence of group norms regarding acceptable safety practices, and ( ) the organization's socialization process for new personnel. safety and patient outcomes can be enhanced by improving or creating organizational characteristics within patient care units, as demonstrated by studies of surgical icus. , each of these factors has a direct bearing on adherence to transmission prevention recommendations. measurement of an institution's culture of safety is useful in designing improvements in health care. , several hospitalbased studies have linked measures of safety culture with both employee adherence to safe practices and reduced exposures to blood and body fluids. [ ] [ ] [ ] [ ] [ ] [ ] [ ] one study of hand hygiene practices concluded that improved adherence requires integration of infection control into the organization's safety culture. several hospitals that are part of the veterans administration health care system have taken specific steps toward improving the safety culture, including error-reporting mechanisms, root cause analyses of identified problems, safety incentives, and employee education. [ ] [ ] [ ] ii.a. . adherence of health care workers to recommended guidelines. hcws' adherence to recommended infection control practices decreases the transmission of infectious agents in health care settings. , , [ ] [ ] [ ] [ ] [ ] several observational studies have shown limited adherence to recommended practices by hcws. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] observed adherence to universal precautions ranged from % to %. , , , , the degree of adherence often depended on the specific practice that was assessed and, for glove use, the circumstance in which the practice was applied. observed rates of appropriate glove use has ranged from a low of % to a high of %. however, % and % adherence with glove use have been reported during arterial blood gas collection and resuscitation, respectively, procedures in which considerable blood contact may occur. , differences in observed adherence have been reported among occupational groups in the same health care facility and between experienced and nonexperienced professionals. in surveys of hcws, self-reported adherence was generally higher than actual adherence found in observational studies. furthermore, where an observational component was included with a self-reported survey, self-perceived adherence was often greater than observed adherence. among nurses and physicians, increasing years of experience is a negative predictor of adherence. , education to improve adherence is the primary intervention that has been studied. whereas positive changes in knowledge and attitude have been demonstrated, , no or only limited accompanying changes in behavior often have been found. , self-reported adherence is higher in groups that received an educational intervention. , in one study, educational interventions that incorporated videotaping and performance feedback were successful in improving adherence during the study period, but the long-term effect of such interventions is not known. the use of videotaping also served to identify system problems (eg, communication and access to ppe) that otherwise may not have been recognized. interest is growing in the use of engineering controls and facility design concepts for improving adherence. whereas the introduction of automated sinks was found to have a negative impact on consistent adherence to handwashing in one study, the use of electronic monitoring and voice prompts to remind hcws to perform hand hygiene and improving accessibility to hand hygiene products increased adherence and contributed to a decrease in hais in another study. more information is needed regarding ways in which technology might improve adherence. improving adherence to infection control practices requires a multifaceted approach that incorporates continuous assessment of both the individual and the work environment. , using several behavioral theories, kretzer and larson concluded that a single intervention (eg, a handwashing campaign or putting up new posters about transmission precautions) likely would be ineffective in improving hcws adherence. improvement requires the organizational leadership to make prevention an institutional priority and integrate infection control practices into the organization's safety culture. a recent review of the literature concluded that variations in organizational factors (eg, safety climate, policies and procedures, education and training) and individual factors (eg, knowledge, perceptions of risk, past experience) were determinants of adherence to infection control guidelines for protection against sars and other respiratory pathogens. surveillance is an essential tool for case finding of single patients or clusters of patients who are infected or colonized with epidemiologically important organisms (eg, susceptible bacteria such as s aureus, s pyogenes [group a streptococcus] or enterobacter-klebsiella spp; mrsa, vre, and other mdros; c difficile; rsv; influenza virus) for which transmission-based precautions may be required. surveillance is defined as the ongoing systematic collection, analysis, interpretation, and dissemination of data regarding a health-related event for use in public health action to reduce morbidity and mortality and to improve health. the work of ignaz semmelweis delineating the role of person-toperson transmission in puerperal sepsis is the earliest example of the use of surveillance data to reduce transmission of infectious agents. surveillance of both process measures and the infection rates to which they are linked is important in evaluating the effectiveness of infection prevention efforts and identifying indications for change. , [ ] [ ] [ ] [ ] the study on the efficacy of nosocomial infection control (senic) found that different combinations of infection control practices resulted in reduced rates of nosocomial surgical site infections, pneumonia, urinary tract infections, and bacteremia in acute care hospitals; however, surveillance was the only component essential for reducing all types of hais. although a similar study has not been conducted in other health care settings, a role for surveillance and the need for novel strategies in ltcfs , , , and in home care [ ] [ ] [ ] [ ] have been described. the essential elements of a surveillance system are ( ) standardized definitions, ( ) identification of patient populations at risk for infection, ( ) statistical analysis (eg, risk adjustment, calculation of rates using appropriate denominators, trend analysis using such methods as statistical process control charts), and ( ) feedback of results to the primary caregivers. [ ] [ ] [ ] [ ] [ ] [ ] data gathered through surveillance of high-risk populations, device use, procedures, and facility locations (eg, icus) are useful in detecting transmission trends. [ ] [ ] [ ] identification of clusters of infections should be followed by a systematic epidemiologic investigation to determine commonalities in persons, places, and time and to guide implementation of interventions and evaluation of the effectiveness of those interventions. targeted surveillance based on the highest-risk areas or patients has been preferred over facility-wide surveillance for the most effective use of resources. , however, for certain epidemiologically important organisms, surveillance may need to be facility-wide. surveillance methods will continue to evolve as health care delivery systems change , and user-friendly electronic tools for electronic tracking and trend analysis become more widely available. , , individuals with experience in health care epidemiology and infection control should be involved in selecting software packages for data aggregation and analysis, to ensure that the need for efficient and accurate hai surveillance will be met. effective surveillance is increasingly important as legislation requiring public reporting of hai rates is passed and states work to develop effective systems to support such legislation. the education and training of hcws is a prerequisite for ensuring that policies and procedures for standard and transmission-based precautions are understood and practiced. understanding the scientific rationale for the precautions will allow hcws to apply procedures correctly, as well as to safely modify precautions based on changing requirements, resources, or health care settings. , , - one study found that the likelihood of hcws developing sars was strongly associated with less than hours of infection control training and poor understanding of infection control procedures. education regarding the important role of vaccines (eg, influenza, measles, varicella, pertussis, pneumococcal) in protecting hcws, their patients, and family members can help improve vaccination rates. [ ] [ ] [ ] [ ] education on the principles and practices for preventing transmission of infectious agents should begin during training in the health professions and be provided to anyone who has an opportunity for contact with patients or medical equipment (eg, nursing and medical staff; therapists and technicians, including respiratory, physical, occupational, radiology, and cardiology personnel; phlebotomists; housekeeping and maintenance staff; and students). in health care facilities, education and training on standard and transmission-based precautions are typically provided at the time of orientation and should be repeated as necessary to maintain competency; updated education and training are necessary when policies and procedures are revised or when a special circumstance occurs, such as an outbreak that requires modification of current practice or adoption of new recommendations. education and training materials and methods appropriate to the hcw's level of responsibility, individual learning habits, and language needs can improve the learning experience. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] education programs for hcws have been associated with sustained improvement in adherence to best practices and a related decrease in device-associated hais in teaching and nonteaching settings , and in medical and surgical icus (coopersmith, # ) . several studies have shown that in addition to targeted education to improve specific practices, periodic assessment and feedback of the hcw's knowledge and adherence to recommended practices are necessary to achieve the desired changes and identify continuing education needs. , [ ] [ ] [ ] [ ] [ ] the effectiveness of this approach for isolation practices has been demonstrated in the control of rsv. , patients, family members, and visitors can be partners in preventing transmission of infections in health care settings. , , - information on standard precautions, especially hand hygiene, respiratory hygiene/cough etiquette, vaccination (especially against influenza), and other routine infection prevention strategies, may be incorporated into patient information materials provided on admission to the health care facility. additional information on transmission-based precautions is best provided when these precautions are initiated. fact sheets, pamphlets, and other printed material may include information on the rationale for the additional precautions, risks to household members, room assignment for transmission-based precautions purposes, explanation of the use of ppe by hcws, and directions for use of such equipment by family members and visitors. such information may be particularly helpful in the home environment, where household members often have the primary responsibility for adherence to recommended infection control practices. hcws must be available and prepared to explain this material and answer questions as needed. hand hygiene has been frequently cited as the single most important practice to reduce the transmission of infectious agents in health care settings , , and is an essential element of standard precautions. the term ''hand hygiene'' includes both handwashing with either plain or antiseptic-containing soap and water and the use of alcohol-based products (gels, rinses, foams) that do not require water. in the absence of visible soiling of hands, approved alcohol-based products for hand disinfection are preferred over antimicrobial or plain soap and water because of their superior microbiocidal activity, reduced drying of the skin, and convenience. have been associated with a sustained decrease in the incidence of mrsa and vre infections primarily in icus. , , [ ] [ ] [ ] [ ] the scientific rationale, indications, methods, and products for hand hygiene have been summarized in previous publications. , the effectiveness of hand hygiene can be reduced by the type and length of fingernails. , , individuals wearing artificial nails have been shown to harbor more pathogenic organisms, especially gram-negative bacilli and yeasts, on the nails and in the subungual area compared with individuals with native nails. , in , the cdc/hicpac recommended (category ia) that artificial fingernails and extenders not be worn by hcws who have contact with high-risk patients (eg, those in icus and operating rooms), due to the association with outbreaks of gram-negative bacillus and candidal infections as confirmed by molecular typing of isolates. , , , [ ] [ ] [ ] [ ] the need to restrict the wearing of artificial fingernails by all hcws who provide direct patient care and those who have contact with other high-risk groups (eg, oncology and cystic fibrosis patients) has not been studied but has been recommended by some experts. currently, such decisions are at the discretion of an individual facility's infection control program. there is less evidence indicating that jewelry affects the quality of hand hygiene. although hand contamination with potential pathogens is increased with ring-wearing, , no studies have related this practice to hcw-to-patient transmission of pathogens. ppe refers to various barriers and respirators used alone or in combination to protect mucous membranes, airways, skin, and clothing from contact with infectious agents. the choice of ppe is based on the nature of the patient interaction and/or the likely mode(s) of transmission. specific guidance on the use of ppe is provided in part iii of this guideline. a suggested procedure for donning and removing ppe aimed at preventing skin or clothing contamination is presented in figure . designated containers for used disposable or reusable ppe should be placed in a location convenient to the site of removal, to facilitate disposal and containment of contaminated materials. hand hygiene is always the final step after removing and disposing of ppe. the following sections highlight the primary uses of and criteria for selecting this equipment. ii.e. . gloves. gloves are used to prevent contamination of hcw hands when ( ) anticipating direct contact with blood or body fluids, mucous membranes, nonintact skin and other potentially infectious material; ( ) having direct contact with patients who are colonized or infected with pathogens transmitted by the contact route (eg, vre, mrsa, rsv , , ); or ( ) handling or touching visibly or potentially contaminated patient care equipment and environmental surfaces. , , gloves can protect both patients and hcws from exposure to infectious material that may be carried on hands. the extent to which gloves will protect hcws from transmission of bloodborne pathogens (eg, hiv, hbv, hcv) after a needlestick or other puncture that penetrates the glove barrier has not yet been determined. although gloves may reduce the volume of blood on the external surface of a sharp by % to %, the residual blood in the lumen of a hollow-bore needle would not be affected; therefore, the effect on transmission risk is unknown. gloves manufactured for health care purposes are subject to fda evaluation and clearance. nonsterile disposable medical gloves made of various materials (eg, latex, vinyl, nitrile) are available for routine patient care. the selection of glove type for nonsurgical use is based on various factors, including the task to be performed, anticipated contact with chemicals and chemotherapeutic agents, latex sensitivity, sizing, and facility policies for creating a latex-free environment. , [ ] [ ] [ ] for contact with blood and body fluids during nonsurgical patient care, a single pair of gloves generally provides adequate barrier protection. however, there is considerable variability among gloves; both the quality of the manufacturing process and type of material influence their barrier effectiveness. whereas there is little difference in the barrier properties of unused intact gloves, studies have shown repeatedly that vinyl gloves have higher failure rates than latex or nitrile gloves when tested under simulated and actual clinical conditions. , [ ] [ ] [ ] [ ] for this reason, either latex or nitrile gloves are preferable for clinical procedures that require manual dexterity or will involve more than brief patient contact. a facility may need to stock gloves in several sizes. heavier, reusable utility gloves are indicated for non-patient care activities, such as handling or cleaning contaminated equipment or surfaces. , , during patient care, transmission of infectious organisms can be reduced by adhering to the principles of working from ''clean'' to ''dirty'' and confining or limiting contamination to those surfaces directly needed for patient care. it may be necessary to change gloves during the care of a single patient to prevent cross-contamination of body sites. , it also may be necessary to change gloves if the patient interaction also involves touching portable computer keyboards or other mobile equipment transported from room to room. discarding gloves between patients is necessary to prevent transmission of infectious material. gloves must not be washed for subsequent reuse, because microorganisms cannot be removed reliably from glove surfaces, and continued glove integrity cannot be ensured. furthermore, glove reuse has been associated with transmission of mrsa and gram-negative bacilli. [ ] [ ] [ ] when gloves are worn in combination with other ppe, they are put on last. gloves that fit snugly around the wrist are preferred for use with an isolation gown, because they will cover the gown cuff and provide a more reliable continuous barrier for the arms, wrists, and hands. proper glove removal will prevent hand contamination (fig ) . hand hygiene after glove removal further ensures that the hands will not carry potentially infectious material that might have penetrated through unrecognized tears or that could have contaminated the hands during glove removal. , , ii.e. . isolation gowns. isolation gowns are used as specified by standard and transmission-based precautions to protect the hcw's arms and exposed body areas and prevent contamination of clothing with blood, body fluids, and other potentially infectious material. , , , [ ] [ ] [ ] the need for and the type of isolation gown selected is based on the nature of the patient interaction, including the anticipated degree of contact with infectious material and potential for blood and body fluid penetration of the barrier. the wearing of isolation gowns and other protective apparel is mandated by the occupational safety and health administration's (osha) bloodborne pathogens standard. clinical and laboratory coats or jackets worn over personal clothing for comfort and/or purposes of identity are not considered ppe. when applying standard precautions, an isolation gown is worn only if contact with blood or body fluid is anticipated. however, when contact precautions are used (ie, to prevent transmission of an infectious agent that is not interrupted by standard precautions alone and is associated with environmental contamination), donning of both gown and gloves on room entry is indicated, to prevent unintentional contact with contaminated environmental surfaces. , , , the routine donning of isolation gowns on entry into an icu or other high-risk area does not prevent or influence potential colonization or infection of patients in those areas, however. , [ ] [ ] [ ] [ ] isolation gowns are always worn in combination with gloves, and with other ppe when indicated. gowns are usually the first piece of ppe to be donned. full coverage of the arms and body front, from neck to the mid-thigh or below, will ensure protection of clothing and exposed upper body areas. several gown sizes should be available in a health care facility to ensure appropriate coverage for staff members. isolation gowns should be removed before leaving the patient care area to prevent possible contamination of the environment outside the patient's room. isolation gowns should be removed in a manner that prevents contamination of clothing or skin (fig ) ; the outer, ''contaminated'' side of the gown is turned inward and rolled into a bundle, and then discarded into a designated container for waste or linen to contain contamination. ii.e. . face protection: masks, goggles, and face shields. ii.e. .a. masks. masks are used for primary purposes in health care settings: ( ) placed on hcws to protect them from contact with infectious material from patients (eg, respiratory secretions and sprays of blood or body fluids), consistent with standard precautions and droplet precautions; ( ) placed on hcws engaged in procedures requiring sterile technique, to protect patients from exposure to infectious agents carried in the hcw's mouth or nose; and ( ) placed on coughing patients to limit potential dissemination of infectious respiratory secretions from the patient to others (ie, respiratory hygiene/cough etiquette). masks may be used in combination with goggles to protect the mouth, nose, and eyes, or, alternatively, a face shield may be used instead of a mask and goggles to provide more complete protection for the face, as discussed below. masks should not be confused with particulate respirators used to prevent inhalation of small particles that may contain infectious agents transmitted through the airborne route, as described below. the mucous membranes of the mouth, nose, and eyes are susceptible portals of entry for infectious agents; other skin surfaces also may be portals if skin integrity is compromised (by, eg, acne, dermatitis). , [ ] [ ] [ ] [ ] therefore, use of ppe to protect these body sites is an important component of standard precautions. the protective effect of masks for exposed hcws has been demonstrated previously. , , , procedures that generate splashes or sprays of blood, body fluids, secretions, or excretions (eg, endotracheal suctioning, bronchoscopy, invasive vascular procedures) require either a face shield (disposable or reusable) or a mask and goggles. [ ] [ ] [ ] [ ] , , , , the wearing of masks, eye protection, and face shields in specified circumstances when blood or body fluid exposure is likely is mandated by osha's bloodborne pathogens standard. appropriate ppe should be selected based on the anticipated level of exposure. two mask types are available for use in health care settings: surgical masks that are cleared by the fda and required to have fluid-resistant properties, and procedure or isolation masks. ,# to date, no studies comparing mask types to determine whether one mask type provides better protection than another have been published. because procedure/isolation masks are not regulated by the fda, they may be more variable in terms of quality and performance than surgical masks. masks come in various shapes (eg, molded and nonmolded), sizes, filtration efficiency, and method of attachment (eg, ties, elastic, ear loops). health care facilities may find that different types of masks are needed to meet individual hcw needs. ii.e. .b. goggles and face shields. guidance on eye protection for infection control has been published. the eye protection chosen for specific work situations (eg, goggles or face shield) depends on the circumstances of exposure, other ppe used, and personal vision needs. personal eyeglasses and contact lenses are not considered adequate eye protection (see http://www.cdc.gov/ niosh/topics/eye/eye-infectious.html). niosh guidelines specify that eye protection must be comfortable, allow for sufficient peripheral vision, and adjustable to ensure a secure fit. a health care facility may need to provide several different types, styles, and sizes of eye protection equipment. indirectly vented goggles with a manufacturer's antifog coating may provide the most reliable practical eye protection from splashes, sprays, and respiratory droplets from multiple angles. newer styles of goggles may provide better indirect airflow properties to reduce fogging, as well as better peripheral vision and more size options for fitting goggles to different workers. many styles of goggles fit adequately over prescription glasses with minimal gaps. although effective as eye protection, goggles do not provide splash or spray protection to other parts of the face. the role of goggles in addition to a mask in preventing exposure to infectious agents transmitted through respiratory droplets has been studied only for rsv. reports published in the mid- s demonstrated that eye protection reduced occupational transmission of rsv. , whether this was due to the prevention hand-eye contact or the prevention of respiratory droplet-eye contact has not been determined. however, subsequent studies demonstrated that rsv transmission is effectively prevented by adherence to standard precautions plus contact precautions and that routine use of goggles is not necessary for this virus. , , , , it is important to remind hcws that even if droplet precautions are not recommended for a specific respiratory tract pathogen, protection for the eyes, nose, and mouth using a mask and goggles or a face shield alone is necessary when a splash or spray of any respiratory secretions or other body fluids is likely to occur, as defined in standard precautions. disposable or nondisposable face shields may be used as an alternative to goggles. compared with goggles, a face shield can provide protection to other facial areas besides the eyes. face shields extending from the chin to crown provide better face and eye protection from splashes and sprays; face shields that wrap around the sides may reduce splashes around the edge of the shield. removal of a face shield, goggles, and mask can be performed safely after gloves have been removed and hand hygiene performed. the ties, earpieces, and/or headband used to secure the equipment to the head are considered ''clean'' and thus safe to touch with bare hands. the front of a mask, goggles, and face shield are considered contaminated (fig ) . ii.e. . respiratory protection. the subject of respiratory protection as it applies to preventing transmission of airborne infectious agents, including the need for and frequency of fit testing is under scientific review and was the subject of a cdc workshop. respiratory protection currently requires the use of a respirator with n or higher-level filtration to prevent inhalation of infectious particles. information about respirators and respiratory protection programs is summarized in the guideline for preventing transmission of mycobacterium tuberculosis in health care settings. respiratory protection is broadly regulated by osha under the general industry standard for respiratory protection ( cfr . ), which requires that us employers in all employment settings implement a program to protect employees from inhalation of toxic materials. osha program components include medical clearance to wear a respirator; provision and use of appropriate respirators, including fit-tested niosh-certified n and higher-level particulate filtering respirators; education on respirator use, and periodic reevaluation of the respiratory protection program. when selecting particulate respirators, models with inherently good fit characteristics (ie, those expected to provide protection factors of $ % to % of wearers) are preferred and theoretically could preclude the need for fit testing. , issues pertaining to respiratory protection remain the subject of ongoing debate. information on various types of respirators is available at http://www.cdc.gov/niosh/ npptl/respirators/respsars.html and in several previously published studies. , , a user-seal check (formerly called a ''fit check'') should be performed by the wearer of a respirator each time that the respirator is donned, to minimize air leakage around the face piece. the optimal frequency of fit testing has not been determined; retesting may be indicated if there is a change in wearer's facial features, onset of a medical condition that would affect respiratory function in the wearer, or a change in the model or size of the respirator that was initially assigned. respiratory protection was first recommended for protection of us hcws from exposure to m tuberculosis in . that recommendation has been maintained in successive revisions of the guidelines for prevention of transmission of tuberculosis in hospitals and other health care settings. , the incremental benefit from respirator use, in addition to administrative and engineering controls (ie, aiirs, early recognition of patients likely to have tuberculosis and prompt placement in an aiir, and maintenance of a patient with suspected tuberculosis in an aiir until no longer infectious), for preventing transmission of airborne infectious agents (eg, m tuberculosis) remains undetermined. although some studies have demonstrated effective prevention of m tuberculosis transmission in hospitals in which surgical masks instead of respirators were used in conjunction with other administrative and engineering controls. , , the cdc currently recommends n or higher-level respirators for personnel exposed to patients with suspected or confirmed tuberculosis. currently, this recommendation also holds for other diseases that could be transmitted through the airborne route, including sars and smallpox, , , until inhalational transmission is better defined or health care-specific ppe more suitable for preventing infection is developed. wearing of respirators is also currently recommended during the performance of aerosol-generating procedures (eg, intubation, bronchoscopy, suctioning) in patients with sars-cov infection, avian influenza, and pandemic influenza (see appendix a). although airborne precautions are recommended for preventing airborne transmission of measles and varicella-zoster viruses, no data are available on which to base a recommendation for respiratory protection to protect susceptible personnel against these infections. transmission of varicella-zoster virus has been prevented among pediatric patients using negativepressure isolation alone. whether respiratory protection (ie, wearing a particulate respirator) will enhance protection from these viruses has not yet been studied. because most hcws have natural or acquired immunity to these viruses, only immune personnel generally care for patients with these infections. [ ] [ ] [ ] [ ] although there is no evidence suggesting that masks are not adequate to protect hcws in these settings, for purposes of consistency and simplicity, or because of difficulties in ascertaining immunity, some facilities may require the use of respirators for entry into all aiirs, regardless of the specific infectious agent present. procedures for safe removal of respirators are provided in figure . in some health care settings, particulate respirators used to provide care for patients with m tuberculosis are reused by the same hcw. this is an acceptable practice providing that the respirator is not damaged or soiled, the fit is not compromised by a change in shape, and the respirator has not been contaminated with blood or body fluids. no data are available on which to base a recommendation regarding the length of time that a respirator may be safely reused. sharps-related injuries. injuries due to needles and other sharps have been associated with transmission of hbv, hcv, and hiv to hcws. , the prevention of sharps injuries has always been an essential element of universal precautions and is now an aspect of standard precautions. , these include measures to handle needles and other sharp devices in a manner that will prevent injury to the user and to others who may encounter the device during or after a procedure. these measures apply to routine patient care and do not address the prevention of sharps injuries and other blood exposures during surgical and other invasive procedures addressed elsewhere. [ ] [ ] [ ] [ ] [ ] since , when osha first issued its bloodborne pathogens standard to protect hcws from blood exposure, the focus of regulatory and legislative activity has been on implementing a hierarchy of control measures. this has included focusing attention on removing sharps hazards through the development and use of engineering controls. the federal needlestick safety and prevention act, signed into law in november , authorized osha's revision of its bloodborne pathogens standard to more explicitly require the use of safety-engineered sharps devices. the cdc has provided guidance on sharps injury prevention, , including guidelines for the design, implementation and evaluation of a comprehensive sharps injury prevention program. ii.f. . prevention of mucous membrane contact. exposure of mucous membranes of the eyes, nose, and mouth to blood and body fluids has been associated with the transmission of bloodborne viruses and other infectious agents to hcws. , , , the prevention of mucous membrane exposures has always been an element of universal precautions and is now an element of standard precautions for routine patient care , and is subject to osha bloodborne pathogen regulations. safe work practices, in addition to wearing ppe, are designed to protect mucous membranes and nonintact skin from contact with potentially infectious material. these include keeping contaminated gloved and ungloved hands from touching the mouth, nose, eyes, or face and positioning patients to direct sprays and splatter away from the caregiver's face. careful placement of ppe before patient contact will help avoid the need to make adjustments to ppe and prevent possible face or mucous membrane contamination during use. in areas where the need for resuscitation is unpredictable, mouthpieces, pocket resuscitation masks with -way valves, and other ventilation devices provide an alternative to mouth-to-mouth resuscitation, preventing exposure of the caregiver's nose and mouth to oral and respiratory fluids during the procedure. ii.f. .a. precautions during aerosol-generating procedures. the performance of procedures that can generate small-particle aerosols (aerosol-generating procedures), such as bronchoscopy, endotracheal intubation, and open suctioning of the respiratory tract, have been associated with transmission of infectious agents to hcws, including m tuberculosis, sars-cov, , , and n meningitidis. protection of the eyes, nose, and mouth, in addition to gown and gloves, is recommended during performance of these procedures in accordance with standard precautions. the use of a particulate respirator is recommended during aerosol-generating procedures when the aerosol is likely to contain m tuberculosis, sars-cov, or avian or pandemic influenza viruses. ii.g. . hospitals and long-term care facilities. options for patient placement include single-patient rooms, -patient rooms, and multibed wards. of these, single-patient rooms are preferred when transmission of an infectious agent is of concern. although some studies have failed to demonstrate the efficacy of single-patient rooms in preventing hais, other published studies, including one commissioned by the aia and the facility guidelines institute, have documented a beneficial relationship between private rooms and reduced infectious and noninfectious adverse patient outcomes. , the aia notes that private rooms are the trend in hospital planning and design. however, most hospitals and ltcfs have multibed rooms and must consider many competing priorities when determining the appropriate room placement for patients (eg, reason for admission; patient characteristics, such as age, gender, and mental status; staffing needs; family requests; psychosocial factors; reimbursement concerns). in the absence of obvious infectious diseases that require specified airborne infection isolation rooms (eg, tuberculosis, sars, chickenpox), the risk of transmission of infectious agents is not always considered when making placement decisions. when only a limited number of single-patient rooms is available, it is prudent to prioritize room assignments for those patients with conditions that facilitate transmission of infectious material to other patients (eg, draining wounds, stool incontinence, uncontained secretions) and those at increased risk of acquisition and adverse outcomes resulting from hais (due to, eg, immunosuppression, open wounds, indwelling catheters, anticipated prolonged length of stay, total dependence on hcws for activities of daily living). , , , , , single-patient rooms are always indicated for patients placed on airborne precautions in a pe and are preferred for patients requiring contact or droplet precautions. , , , , , during a suspected or proven outbreak caused by a pathogen whose reservoir is the gastrointestinal tract, the use of single-patient rooms with private bathrooms limits opportunities for transmission, especially when the colonized or infected patient has poor personal hygiene habits or fecal incontinence, or cannot be expected to assist in maintaining procedures that prevent transmission of microorganisms (eg, infants, children, and patients with altered mental status or developmental delay). in the absence of continued transmission, it is not necessary to provide a private bathroom for patients colonized or infected with enteric pathogens as long as personal hygiene practices and standard precautions (especially hand hygiene and appropriate environmental cleaning) are maintained. assignment of a dedicated commode to a patient, and cleaning and disinfecting fixtures and equipment that may have fecal contamination (eg, bathrooms, commodes, scales used for weighing diapers) and the adjacent surfaces with appropriate agents may be especially important when a single-patient room cannot be assigned, because environmental contamination with intestinal tract pathogens is likely from both continent and incontinent patients. , the results of several studies that investigated the benefit of a single-patient room in preventing transmission of c difficile were inconclusive. , [ ] [ ] [ ] some studies have shown that being in the same room with a colonized or infected patient is not necessarily a risk factor for transmission; , - however, for children, the risk of health care-associated diarrhea is increased with the increased number of patients per room. these findings demonstrate that patient factors are important determinants of infection transmission risks. the need for a single-patient room and/or private bathroom for any patient is best determined on a case-by-case basis. cohorting is the practice of grouping together patients who are colonized or infected with the same organism to confine their care to a single area and prevent contact with other patients. cohorts are created based on clinical diagnosis, microbiologic confirmation (when available), epidemiology, and mode of transmission of the infectious agent. avoiding placing severely immunosuppressed patients in rooms with other patients is generally preferred. cohorting has been extensively used for managing outbreaks of mdros, including mrsa, rotavirus, and sars. modeling studies provide additional support for cohorting patients to control outbreaks; - however, cohorting often is implemented only after routine infection control measures have failed to control an outbreak. assigning or cohorting hcws to care only for patients infected or colonized with a single target pathogen limits further transmission of the target pathogen to uninfected patients, , but is difficult to achieve in the face of current staffing shortages in hospitals and residential health care sites. [ ] [ ] [ ] however, cohorting of hcws may be beneficial when transmission continues after implementing routine infection control measures and creating patient cohorts. during periods when rsv, human metapneumovirus, parainfluenza, influenza, other respiratory viruses, and rotavirus are circulating in the community, cohorting based on the presenting clinical syndrome is often a priority in facilities that care for infants and young children. for example, during the respiratory virus season, infants may be cohorted based solely on the clinical diagnosis of bronchiolitis, due to the logistical difficulties and costs associated with requiring microbiologic confirmation before room placement and the predominance of rsv during most of the season. however, when available, single-patient rooms are always preferred, because a common clinical presentation (eg, bronchiolitis), can be caused by more than infectious agent. , , furthermore, the inability of infants and children to contain body fluids, and the close physical contact associated with their care, increases the risk of infection transmission for patients and personnel in this setting. , ii.g. . ambulatory care settings. patients actively infected with or incubating transmissible infectious diseases are frequently seen in ambulatory settings (eg, outpatient clinics, physicians' offices, emergency departments) and potentially expose hcws and other patients, family members, and visitors. , , , , , in response to the global outbreak of sars in and in preparation for pandemic influenza, hcws working in outpatient settings are urged to implement source containment measures (eg, asking coughing patients to wear a surgical mask or cover coughing with tissues) to prevent transmission of respiratory infections, beginning at the initial patient encounter, , , as described in section iii.a. .a. signs can be posted at the facility's entrance or at the reception or registration desk requesting that the patient or individuals accompanying the patient promptly inform the receptionist of any symptoms of respiratory infection (eg, cough, flulike illness, increased production of respiratory secretions). the presence of diarrhea, skin rash, or known or suspected exposure to a transmissible disease (eg, measles, pertussis, chickenpox, tuberculosis) also could be added. prompt placement of a potentially infectious patient in an examination room limits the number of exposed individuals in the common waiting area. in waiting areas, maintaining a distance between symptomatic and nonsymptomatic patients (eg, . feet), in addition to source control measures, may limit exposures. however, infections transmitted through the airborne route (eg, m tuberculosis, measles, chickenpox) require additional precautions. , , patients suspected of having such an infection can wear a surgical mask for source containment, if tolerated, and should be placed in an examination room (preferably an aiir) as soon as possible. if this is not possible, then having the patient wear a mask and segregating the patient from other patients in the waiting area will reduce the risk of exposing others. because the person(s) accompanying the patient also may be infectious, application of the same infection control precautions may be extended to these persons if they are symptomatic. , , family members accompanying children admitted with suspected m tuberculosis have been found to have unsuspected pulmonary tuberculosis with cavitary lesions, even when asymptomatic. , patients with underlying conditions that increase their susceptibility to infection (eg, immunocompromised status , or cystic fibrosis ) require special efforts to protect them from exposure to infected patients in common waiting areas. informing the receptionist of their infection risk on arrival allows appropriate steps to further protect these patients from infection. in some cystic fibrosis clinics, to avoid exposure to other patients who could be colonized with b cepacia, patients have been given beepers on registration so that they may leave the area and receive notification to return when an examination room becomes available. ii.g. . home care. in home care, patient placement concerns focus on protecting others in the home from exposure to an infectious household member. for individuals who are especially vulnerable to adverse outcomes associated with certain infections, it may be beneficial to either remove them from the home or segregate them within the home. persons who are not part of the household may need to be prohibited from visiting during the period of infectivity. for example, in a situation where a patient with pulmonary tuberculosis is contagious and being cared for at home, very young children (age under years) and immunocompromised persons who have not yet been infected should be removed or excluded from the household. during the sars outbreak of , segregation of infected persons during the communicable phase of the illness was found to be beneficial in preventing household transmission. , several principles guide the transport of patients requiring transmission-based precautions. in the inpatient and residential settings, these include the following: . limiting transport of such patients to essential purposes, such as diagnostic and therapeutic procedures that cannot be performed in the patient's room. . when transport is necessary, applying appropriate barriers on the patient (eg, mask, gown, wrapping in sheets or use of impervious dressings to cover the affected areas) when infectious skin lesions or drainage are present, consistent with the route and risk of transmission. . notifying hcws in the receiving area of the patient's impending arrival and of the necessary precautions to prevent transmission. . for patients being transported outside the facility, informing the receiving facility and the medi-van or emergency vehicle personnel in advance about the type of transmission-based precautions being used. for tuberculosis, additional precautions may be needed in a small shared air space, such as in an ambulance. cleaning and disinfecting noncritical surfaces in patient care areas is an aspect of standard precautions. in general, these procedures do not need to be changed for patients on transmission-based precautions. the cleaning and disinfection of all patient care areas is important for frequently touched surfaces, especially those closest to the patient, which are most likely to be contaminated (eg, bedrails, bedside tables, commodes, doorknobs, sinks, surfaces and equipment in close proximity to the patient). , , , the frequency or intensity of cleaning may need to be changed, based on the patient's level of hygiene and the degree of environmental contamination and for certain infectious agents with reservoirs in the intestinal tract. this may be particularly important in ltcfs and pediatric facilities, where patients with stool and urine incontinence are encountered more frequently. in addition, increased frequency of cleaning may be needed in a pe to minimize dust accumulation. special recommendations for cleaning and disinfecting environmental surfaces in dialysis centers have been published previously. in all health care settings, administrative, staffing, and scheduling activities should prioritize the proper cleaning and disinfection of surfaces that could be implicated in transmission. during a suspected or proven outbreak in which an environmental reservoir is suspected, routine cleaning procedures should be reviewed, and the need for additional trained cleaning staff should be assessed. adherence should be monitored and reinforced to promote consistent and correct cleaning. us environmental protection agency-registered disinfectants or detergents/disinfectants that best meet the overall needs of the health care facility for routine cleaning and disinfection should be selected. , in general, use of the existing facility detergent/disinfectant according to the manufacturer's recommendations for amount, dilution, and contact time is sufficient to remove pathogens from surfaces of rooms where colonized or infected individuals were housed. this includes those pathogens that are resistant to multiple classes of antimicrobial agents (eg, c difficile, vre, mrsa, mdr-gnb , , , , , , ). most often, environmental reservoirs of pathogens during outbreaks are related to a failure to follow recommended procedures for cleaning and disinfection, rather than to the specific cleaning and disinfectant agents used. [ ] [ ] [ ] [ ] certain pathogens (eg, rotavirus, noroviruses, c difficile) may be resistant to some routinely used hospital disinfectants. , , [ ] [ ] [ ] [ ] [ ] [ ] the role of specific disinfectants in limiting transmission of rotavirus has been demonstrated experimentally. also, because c difficile may display increased levels of spore production when exposed to non-chlorine-based cleaning agents, and because these spores are more resistant than vegetative cells to commonly used surface disinfectants, some investigators have recommended the use of a : dilution of . % sodium hypochlorite (household bleach) and water for routine environmental disinfection of rooms of patients with c difficile when there is continued transmission. , one study found an association between the use of a hypochlorite solution and decreased rates of c difficile infections. the need to change disinfectants based on the presence of these organisms can be determined in consultation with the infection control committee. , , detailed recommendations for disinfection and sterilization of surfaces and medical equipment that have been in contact with prion-containing tissue or high risk body fluids, and for cleaning of blood and body substance spills, are available in the guidelines for environmental infection control in health care facilities and in the guideline for disinfection and sterilization. medical equipment and instruments/devices must be cleaned and maintained according to the manufacturers' instructions to prevent patient-to-patient transmission of infectious agents. , , , cleaning to remove organic material always must precede highlevel disinfection and sterilization of critical and semicritical instruments and devices, because residual proteinacous material reduces the effectiveness of the disinfection and sterilization processes. , noncritical equipment, such as commodes, intravenous pumps, and ventilators, must be thoroughly cleaned and disinfected before being used on another patient. all such equipment and devices should be handled in a manner that will prevent hcw and environmental contact with potentially infectious material. it is important to include computers and personal digital assistants used in patient care in policies for cleaning and disinfection of noncritical items. the literature on contamination of computers with pathogens has been summarized, and reports have linked computer contamination to colonization and infections in patients. , although keyboard covers and washable keyboards that can be easily disinfected are available, the infection control benefit of these items and their optimal management have not yet been determined. in all health care settings, providing patients who are on transmission-based precautions with dedicated noncritical medical equipment (eg, stethoscope, blood pressure cuff, electronic thermometer) has proven beneficial for preventing transmission. , , , , when this is not possible, disinfection of this equipment after each use is recommended. other previously published guidelines should be consulted for detailed guidance in developing specific protocols for cleaning and reprocessing medical equipment and patient care items in both routine and special circumstances. , , , , , , in home care, it is preferable to remove visible blood or body fluids from durable medical equipment before it leaves the home. equipment can be cleaned onsite using a detergent/disinfectant and, when possible, should be placed in a plastic bag for transport to the reprocessing location. , although soiled textiles, including bedding, towels, and patient or resident clothing, may be contaminated with pathogenic microorganisms, the risk of disease transmission is negligible if these textiles are handled, transported, and laundered in a safe manner. , , key principles for handling soiled laundry are ( ) avoiding shaking the items or handling them in any way that may aerosolize infectious agents, ( ) avoiding contact of one's body and personal clothing with the soiled items being handled, and ( ) containing soiled items in a laundry bag or designated bin. if a laundry chute is used, it must be maintained to minimize dispersion of aerosols from contaminated items. methods of handling, transporting, and laundering soiled textiles are determined by organizational policy and any applicable regulations; guidance is provided in the guidelines for environmental infection control in health care facilities. rather than rigid rules and regulations, hygienic and common sense storage and processing of clean textiles is recommended. , when laundering is done outside of a health care facility, the clean items must be packaged or completely covered and placed in an enclosed space during transport to prevent contamination with outside air or construction dust that could contain infectious fungal spores that pose a risk for immunocompromised patients. institutions are required to launder garments used as ppe and uniforms visibly soiled with blood or infective material. little data exist on the safety of home laundering of hcw uniforms, but no increase in infection rates was observed in the one published study, and no pathogens were recovered from home-or hospital-laundered scrubs in another study. in the home, textiles and laundry from patients with potentially transmissible infectious pathogens do not require special handling or separate laundering and may be washed with warm water and detergent. , , the management of solid waste emanating from the health care environment is subject to federal and state regulations for medical and nonmedical waste. , no additional precautions are needed for nonmedical solid waste removed from rooms of patients on transmission-based precautions. solid waste may be contained in a single bag of sufficient strength. the combination of hot water and detergents used in dishwashers is sufficient to decontaminate dishware and eating utensils. therefore, no special precautions are needed for dishware (eg, dishes, glasses, cups) or eating utensils. reusable dishware and utensils may be used for patients requiring transmission-based precautions. in the home and other communal settings, eating utensils and drinking vessels should not be shared, consistent with principles of good personal hygiene and to help prevent transmission of respiratory viruses, herpes simplex virus, and infectious agents that infect the gastrointestinal tract and are transmitted by the fecal/oral route (eg, hepatitis a virus, noroviruses). if adequate resources for cleaning utensils and dishes are not available, then disposable products may be used. important adjunctive measures that are not considered primary components of programs to prevent transmission of infectious agents but nonetheless improve the effectiveness of such programs include ( ) antimicrobial management programs, ( ) postexposure chemoprophylaxis with antiviral or antibacterial agents, ( ) vaccines used both for pre-exposure and postexposure prevention, and ( ) screening and restricting visitors with signs of transmissible infections. detailed discussion of judicious use of antimicrobial agents is beyond the scope of this document; however, this topic has been addressed in a previous cdc guideline (http://www.cdc.gov/ncidod/dhqp/pdf/ar/ mdroguideline .pdf). ii.n. . chemoprophylaxis. antimicrobial agents and topical antiseptics may be used to prevent infection and potential outbreaks of selected agents. infections for which postexposure chemoprophylaxis is recommended under defined conditions include b pertussis, , n meningitides, b anthracis after environmental exposure to aeosolizable material, influenza virus, hiv, and group a streptococcus. orally administered antimicrobials also may be used under defined circumstances for mrsa decolonization of patients or hcws. another form of chemoprophylaxis involves the use of topical antiseptic agents. for example, triple dye is routinely used on the umbilical cords of term newborns to reduce the risk of colonization, skin infections, and omphalitis caused by s aureus, including mrsa, and group a streptococcus. , extension of the use of triple dye to low birth weight infants in a nicu was one component of a program that controlled a long-standing mrsa outbreak. topical antiseptics (eg, mupirocin) also are used for decolonization of hcws or selected patients colonized with mrsa, as discussed in the mdro guideline , [ ] [ ] [ ] [ ] ii.n. . immunoprophylaxis. certain immunizations recommended for susceptible hcws have decreased the risk of infection and the potential for transmission in health care facilities. , the osha mandate requiring employers to offer hbv vaccination to hcws has played a substantial role in the sharp decline in incidence of occupational hbv infection. , the routine administration of varicella vaccine to hcws has decreased the need to place susceptible hcws on administrative leave after exposure to patients with varicella. in addition, reports of health care-associated transmission of rubella in obstetric clinics , and measles in acute care settings demonstrate the importance of immunization of susceptible hcws against childhood diseases. many states have requirements for vaccination of hcws for measles and rubella in the absence of evidence of immunity. annual influenza vaccine campaigns targeted at patients and hcws in ltcfs and acute care settings have been instrumental in preventing or limiting institutional outbreaks; consequently, increasing attention is being directed toward improving influenza vaccination rates in hcws. , , , [ ] [ ] [ ] transmission of b pertussis in health care facilities has been associated with large and costly outbreaks that include both hcws and patients. , , , , , , , hcws in close contact with infants with pertussis are at particularly high risk because of waning immunity and, until , the absence of a vaccine appropriate for adults. but acellular pertussis vaccines were licensed in the united states in , for use in individuals age to years and the other for use in those age to years. current advisory committee on immunization practices provisional recommendations include immunization of adolescents and adults, especially those in contact with infants under age months and hcws with direct patient contact. , immunization of children and adults will help prevent the introduction of vaccine-preventable diseases into health care settings. the recommended immunization schedule for children is published annually in the january issues of the morbidity and mortality weekly report, with interim updates as needed. , an adult immunization schedule also is available for healthy adults and those with special immunization needs due to high-risk medical conditions. some vaccines are also used for postexposure prophylaxis of susceptible individuals, including varicella, influenza, hepatitis b, and smallpox vaccines. , in the future, administration of a newly developed s aureus conjugate vaccine (still under investigation) to selected patients may provide a novel method of preventing health care-associated s aureus (including mrsa) infections in high-risk groups (eg, hemodialysis patients and candidates for selected surgical procedures). , immune globulin preparations also are used for postexposure prophylaxis of certain infectious agents under specified circumstances (eg, varicella-zoster virus, hbv, rabies, measles and hepatitis a virus , , ). the rsv monoclonal antibody preparation palivizumab may have contributed to controlling a nosocomial outbreak of rsv in one nicu, but there is insufficient evidence to support a routine recommendation for its use in this setting. ii.n. , , , and sars , [ ] [ ] [ ] . effective methods for visitor screening in health care settings have not yet been studied, however. visitor screening is especially important during community outbreaks of infectious diseases and for high-risk patient units. sibling visits are often encouraged in birthing centers, postpartum rooms, pediatric inpatient units, picus, and residential settings for children; in hospital settings, a child visitor should visit only his or her own sibling. screening of visiting siblings and other children before they are allowed into clinical areas is necessary to prevent the introduction of childhood illnesses and common respiratory infections. screening may be passive, through the use of signs to alert family members and visitors with signs and symptoms of communicable diseases not to enter clinical areas. more active screening may include the completion of a screening tool or questionnaire to elicit information related to recent exposures or current symptoms. this information is reviewed by the facility staff, after which the visitor is either permitted to visit or is excluded. family and household members visiting pediatric patients with pertussis and tuberculosis may need to be screened for a history of exposure, as well as signs and symptoms of current infection. potentially infectious visitors are excluded until they receive appropriate medical screening, diagnosis, or treatment. if exclusion is not considered to be in the best interest of the patient or family (ie, primary family members of critically or terminally ill patients), then the symptomatic visitor must wear a mask while in the health care facility and remain in the patient's room, avoiding exposure to others, especially in public waiting areas and the cafeteria. visitor screening is used consistently on hsct units. , however, considering the experience during the sars outbreaks and the potential for pandemic influenza, developing effective visitor screening systems will be beneficial. education concerning respiratory hygiene/cough etiquette is a useful adjunct to visitor screening. ii.n. .b. use of barrier precautions by visitors. the use of gowns, gloves, and masks by visitors in health care settings has not been addressed specifically in the scientific literature. some studies included the use of gowns and gloves by visitors in the control of mdros but did not perform a separate analysis to determine whether their use by visitors had a measurable impact. [ ] [ ] [ ] family members or visitors who are providing care to or otherwise are in very close contact with the patient (eg, feeding, holding) may also have contact with other patients and could contribute to transmission in the absence of effective barrier precautions. specific recommendations may vary by facility or by unit and should be determined by the specific level of interaction. there are tiers of hicpac/cdc precautions to prevent transmission of infectious agents, standard precautions and transmission-based precautions. standard precautions are intended to be applied to the care of all patients in all health care settings, regardless of the suspected or confirmed presence of an infectious agent. implementation of standard precautions constitutes the primary strategy for the prevention of health care-associated transmission of infectious agents among patients and hcws. transmission-based precautions are for patients who are known or suspected to be infected or colonized with infectious agents, including certain epidemiologically important pathogens, which require additional control measures to effectively prevent transmission. because the infecting agent often is not known at the time of admission to a health care facility, transmission-based precautions are used empirically, according to the clinical syndrome and the likely etiologic agents at the time, and then modified when the pathogen is identified or a transmissible infectious etiology is ruled out. examples of this syndromic approach are presented in table . the hicpac/cdc guidelines also include recommendations for creating a protective environment for allogeneic hsct patients. the specific elements of standard and transmission-based precautions are discussed in part ii of this guideline. in part iii, the circumstances in which standard precautions, transmission-based precautions, and a protective environment are applied are discussed. tables and summarize the key elements of these sets of precautions standard precautions combine the major features of universal precautions , and body substance isolation and are based on the principle that all blood, body fluids, secretions, excretions except sweat, nonintact skin, and mucous membranes may contain transmissible infectious agents. standard precautions include a group of infection prevention practices that apply to all patients, regardless of suspected or confirmed infection status, in any setting in which health care is delivered (table ). these include hand hygiene; use of gloves, gown, mask, eye protection, or face shield, depending on the anticipated exposure; and safe injection practices. also, equipment or items in the patient environment likely to have been contaminated with infectious body fluids must be handled in a manner to prevent transmission of infectious agents (eg, wear gloves for direct contact, contain heavily soiled equipment, properly clean and disinfect or sterilize reusable equipment before use on another patient). the application of standard precautions during patient care is determined by the nature of the hcw-patient interaction and the extent of anticipated blood, body fluid, or pathogen exposure. for some interactions (eg, performing venipuncture), only gloves may be needed; during other interactions (eg, intubation), use of gloves, gown, and face shield or mask and goggles is necessary. education and training on the principles and rationale for recommended practices are critical elements of standard precautions because they facilitate appropriate decision-making and promote adherence when hcws are faced with new circumstances. , [ ] [ ] [ ] [ ] [ ] [ ] an example of the importance of the use of standard precautions is intubation, especially under emergency circumstances when infectious agents may not be suspected, but later are identified (eg, sars-cov, n meningitides). the application of standard precautions is described below and summarized in table . guidance on donning and removing gloves, gowns and other ppe is presented in figure . standard precautions are also intended to protect patients by ensuring that hcws do not carry infectious agents to patients on their hands or via equipment used during patient care. , , the strategy proposed has been termed respiratory hygiene/cough etiquette , and is intended to be incorporated into infection control practices as a new component of standard precautions. the strategy is targeted at patients and accompanying family members and friends with undiagnosed transmissible respiratory infections, and applies to any person with signs of illness including cough, congestion, rhinorrhea, or increased production of respiratory secretions when entering a health care facility. , , the term cough etiquette is derived from recommended source control measures for m tuberculosis. , the elements of respiratory hygiene/cough etiquette include ( ) education of health care facility staff, patients, and visitors; ( ) posted signs, in language(s) appropriate to the population served, with instructions to patients and accompanying family members or friends; ( ) source control measures (eg, covering the mouth/nose with a tissue when coughing and prompt disposal of used tissues, using surgical masks on the coughing person when tolerated and appropriate); ( ) hand hygiene after contact with respiratory secretions; and ( ) spatial separation, ideally . feet, of persons with respiratory infections in common waiting areas when possible. covering sneezes and coughs and placing masks on coughing patients are proven means of source containment that prevent infected persons from dispersing respiratory secretions into the air. , , , masking may be difficult in some settings, (eg, pediatrics), in which case the emphasis by necessity may be on cough etiquette. physical proximity of , feet has been associated with an increased risk for transmission of infections through the droplet route (eg, n meningitidis and group a streptococcus ) and thus supports the practice of distancing infected persons from others who are not infected. the effectiveness of good hygiene practices, especially hand hygiene, in preventing transmission of viruses and reducing the incidence of respiratory infections both within and outside [ ] [ ] [ ] health care settings is summarized in several reviews. , , these measures should be effective in decreasing the risk of transmission of pathogens contained in large respiratory droplets (eg, influenza virus, adenovirus, b pertussis, and m pneumoniae ). although fever will be present in many respiratory infections, patients with pertussis and mild upper respiratory tract infections are often afebrile. therefore, the absence of fever does not always exclude a respiratory infection. patients who have asthma, allergic rhinitis, or chronic obstructive lung disease also may be coughing and sneezing. although these patients often are not infectious, cough etiquette measures are prudent. hcws are advised to observe droplet precautions (ie, wear a mask) and hand hygiene when examining and caring for patients with signs and symptoms of a respiratory infection. hcws who have a respiratory infection are advised to avoid direct patient contact, especially with high-risk patients. if this is not possible, then a mask should be worn while providing patient care. iii.a. .b. safe injection practices. the investigation of large outbreaks of hbv and hcv among patients in ambulatory care facilities in the united states identified a need to define and reinforce safe injection practices. the outbreaks occurred in a private medical practice, a pain clinic, an endoscopy clinic, and a hematology/oncology clinic. the primary breaches in infection control practice that contributed to these outbreaks were reinsertion of used needles into a multiple-dose vial or solution container (eg, saline bag) and use of a single needle/syringe to administer intravenous medication to multiple patients. in of these outbreaks, preparation of medications in the same workspace where used needle/syringes were dismantled also may have been a contributing factor. these and other outbreaks of viral hepatitis could have been prevented by adherence to basic principles of aseptic technique for the preparation and administration of parenteral medications. , these include the use of a sterile, single-use, disposable needle and syringe for each injection given and prevention of contamination of injection equipment and medication. whenever possible, use of single-dose vials is preferred over multiple-dose vials, especially when medications will be administered to multiple patients. outbreaks related to unsafe injection practices indicate that some hcws are unaware of, do not understand, or do not adhere to basic principles of infection control and aseptic technique. a survey of us health care workers who provide medication through injection found that % to % reused the same needle and/or syringe on multiple patients. among the deficiencies identified in recent outbreaks were a lack of oversight of personnel and failure to follow up on reported breaches in infection control practices in ambulatory settings. therefore, to ensure that all hcws understand and adhere to recommended practices, principles of infection control and aseptic technique need to be reinforced in training programs and incorporated into institutional polices that are monitored for adherence. iii.a. .c. infection control practices for special lumbar puncture procedures. in , the cdc investigated cases of postmyelography meningitis that either were reported to the cdc or identified through a survey of the emerging infections network of the infectious disease society of america. blood and/or cerebrospinal fluid of all cases yielded streptococcal species consistent with oropharyngeal flora and there were changes in the csf indices and clinical status indicative of bacterial meningitis. equipment and products used during these procedures (eg, contrast media) were excluded as probable sources of contamination. procedural details available for cases determined that antiseptic skin preparations and sterile gloves had been used. however, none of the clinicians wore a face mask, giving rise to the speculation that droplet transmission of oralpharyngeal flora was the most likely explanation for these infections. bacterial meningitis after myelography and other spinal procedures (eg, lumbar puncture, spinal and epidural anesthesia, intrathecal chemotherapy) has been reported previously. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as a result, the question of whether face masks should be worn to prevent droplet spread of oral flora during spinal procedures (eg, myelography, lumbar puncture, spinal anesthesia) has been debated. , face masks are effective in limiting the dispersal of oropharyngeal droplets and are recommended for the placement of central venous catheters. in october , hicpac reviewed the evidence and concluded that there is sufficient experience to warrant the additional protection of a face mask for the individual placing a catheter or injecting material into the spinal or epidural space. there are categories of transmission-based precautions: contact precautions, droplet precautions, and airborne precautions. transmission-based precautions are used when the route(s) of transmission is (are) not completely interrupted using standard precautions alone. for some diseases that have multiple routes of transmission (eg, sars), more than transmission-based precautions category may be used. when used either singly or in combination, they are always used in addition to standard precautions. see appendix a for recommended precautions for specific infections. when transmission-based precautions are indicated, efforts must be made to counteract possible adverse effects on patients (ie, anxiety, depression and other mood disturbances, - perceptions of stigma, reduced contact with clinical staff, [ ] [ ] [ ] and increases in preventable adverse events ) to improve acceptance by the patients and adherence by hcws. iii.b. . contact precautions. contact precautions are intended to prevent transmission of infectious agents, including epidemiologically important microorganisms, which are spread by direct or indirect contact with the patient or the patient's environment as described in section i.b. .a. the specific agents and circumstance for which contact precautions are indicated are found in appendix a. the application of contact precautions for patients infected or colonized with mdros is described in the hicpac/cdc mdro guideline. contact precautions also apply where the presence of excessive wound drainage, fecal incontinence, or other discharges from the body suggest an increased potential for extensive environmental contamination and risk of transmission. a single-patient room is preferred for patients who require contact precautions. when a single-patient room is not available, consultation with infection control personnel is recommended to assess the various risks associated with other patient placement options (eg, cohorting, keeping the patient with an existing roommate). in multipatient rooms, $ feet spatial separation between beds is advised to reduce the opportunities for inadvertent sharing of items between the infected/colonized patient and other patients. hcws caring for patients on contact precautions wear a gown and gloves for all interactions that may involve contact with the patient or potentially contaminated areas in the patient's environment. donning ppe on room entry and discarding before exiting the patient room is done to contain pathogens, especially those that have been implicated in transmission through environmental contamination (eg, vre, c difficile, noroviruses and other intestinal tract pathogens, rsv). , , , , , , iii.b. . droplet precautions. droplet precautions are intended to prevent transmission of pathogens spread through close respiratory or mucous membrane contact with respiratory secretions as described in section i.b. .b. because these pathogens do not remain infectious over long distances in a health care facility, special air handling and ventilation are not required to prevent droplet transmission. infectious agents for which droplet precautions are indicated are listed in appendix a and include b pertussis, influenza virus, adenovirus, rhinovirus, n meningitides, and group a streptococcus (for the first hours of antimicrobial therapy). a single-patient room is preferred for patients who require droplet precautions. when a single-patient room is not available, consultation with infection control personnel is recommended to assess the various risks associated with other patient placement options (eg, cohorting, keeping the patient with an existing roommate). spatial separation of $ feet and drawing the curtain between patient beds is especially important for patients in multibed rooms with infections transmitted by the droplet route. hcws wear a mask (a respirator is not necessary) for close contact with infectious patient; the mask is generally donned on room entry. patients on droplet precautions who must be transported outside of the room should wear a mask if tolerated and follow respiratory hygiene/cough etiquette. iii.b. . airborne precautions. airborne precautions prevent transmission of infectious agents that remain infectious over long distances when suspended in the air (eg, rubeola virus [measles], varicella virus [chickenpox], m tuberculosis, and possibly sars-cov), as described in section i.b. .c and appendix a. the preferred placement for patients who require airborne precautions is in an aiir, a single-patient room equipped with special air handling and ventilation capacity that meet the aia/facility guidelines institute standards for aiirs (ie, monitored negative pressure relative to the surrounding area; air exchanges per hour for new construction and renovation and air exchanges per hour for existing facilities; air exhausted directly to the outside or recirculated through hepa filtration before return). , some states require the availability of such rooms in hospitals, emergency departments, and nursing homes that care for patients with m tuberculosis. a respiratory protection program that includes education about use of respirators, fit testing, and user seal checks is required in any facility with aiirs. in settings where airborne precautions cannot be implemented due to limited engineering resources (eg, physician offices), masking the patient, placing the patient in a private room (eg, office examination room) with the door closed, and providing n or higher-level respirators or masks if respirators are not available for hcws will reduce the likelihood of airborne transmission until the patient is either transferred to a facility with an aiir or returned to the home environment, as deemed medically appropriate. hcws caring for patients on airborne precautions wear a mask or respirator, depending on the disease-specific recommendations (see section ii.e. , table , and appendix a), that is donned before room entry. whenever possible, nonimmune hcws should not care for patients with vaccine-preventable airborne diseases (eg, measles, chickenpox, smallpox). diagnosis of many infections requires laboratory confirmation. because laboratory tests, especially those that depend on culture techniques, often require or more days for completion, transmission-based precautions must be implemented while test results are pending, based on the clinical presentation and likely pathogens. use of appropriate transmission-based precautions at the time a patient develops symptoms or signs of transmissible infection, or arrives at a health care facility for care, reduces transmission opportunities. although it is not possible to identify prospectively all patients needing transmission-based precautions, certain clinical syndromes and conditions carry a sufficiently high risk to warrant their use empirically while confirmatory tests are pending (see table ). icps are encouraged to modify or adapt this table according to local conditions. transmission-based precautions remain in effect for limited periods (ie, while the risk for transmission of the infectious agent persists or for the duration of the illness (see appendix a). for most infectious diseases, this duration reflects known patterns of persistence and shedding of infectious agents associated with the natural history of the infectious process and its treatment. for some diseases (eg, pharyngeal or cutaneous diphtheria, rsv), transmission-based precautions remain in effect until culture or antigen-detection test results document eradication of the pathogen and, for rsv, symptomatic disease is resolved. for other diseases (eg, m tuberculosis), state laws and regulations and health care facility policies may dictate the duration of precautions. in immunocompromised patients, viral shedding can persist for prolonged periods of time (many weeks to months) and transmission to others may occur during that time; therefore, the duration of contact and/or droplet precautions may be prolonged for many weeks. , [ ] [ ] [ ] [ ] [ ] [ ] the duration of contact precautions for patients who are colonized or infected with mdros remains undefined. mrsa is the only mdro for which effective decolonization regimens are available. however, carriers of mrsa who have negative nasal cultures after a course of systemic or topical therapy may resume shedding mrsa in the weeks after therapy. , although early guidelines for vre suggested discontinuation of contact precautions after stool cultures obtained at weekly intervals proved negative, subsequent experiences have indicated that such screening may fail to detect colonization that can persist for . year. , [ ] [ ] [ ] likewise, available data indicate that colonization with vre, mrsa, and possibly mdr-gnb can persist for many months, especially in the presence of severe underlying disease, invasive devices, and recurrent courses of antimicrobial agents. it may be prudent to assume that mdro carriers are colonized permanently and manage them accordingly. alternatively, an interval free of hospitalizations, antimicrobial therapy, and invasive devices (eg, or months) before reculturing patients to document clearance of carriage may be used. determination of the best strategy awaits the results of additional studies. see the hicpac/cdc mdro guideline for a discussion of possible criteria to discontinue contact precautions for patients colonized or infected with mdros. although transmission-based precautions generally apply in all health care settings, exceptions exist. for example, in home care, aiirs are not available. furthermore, family members already exposed to diseases such as varicella and tuberculosis would not use masks or respiratory protection, but visiting hcws would need to use such protection. similarly, management of patients colonized or infected with mdros may necessitate contact precautions in acute care hospitals and in some ltcfs when there is continued transmission, but the risk of transmission in ambulatory care and home care has not been defined. consistent use of standard precautions may suffice in these settings, but more information is needed. a pe is designed for allogeneic hsct patients to minimize fungal spore counts in the air and reduce the risk of invasive environmental fungal infections (see table for specifications). , [ ] [ ] [ ] the need for such controls has been demonstrated in studies of aspergillosis outbreaks associated with construction. , , , , as defined by the aia and presented in detail in the cdc's guideline for environmental infection control in health care facilities, , air quality for hsct patients is improved through a combination of environmental controls that include ( ) hepa filtration of incoming air, ( ) directed room air flow, ( ) positive room air pressure relative to the corridor, ( ) well-sealed rooms (including sealed walls, floors, ceilings, windows, electrical outlets) to prevent flow of air from the outside, ( ) ventilation to provide $ air changes per hour, ( ) strategies to minimize dust (eg, scrubbable surfaces rather than upholstery and carpet, and routinely cleaning crevices and sprinkler heads), and ( ) prohibiting dried and fresh flowers and potted plants in the rooms of hsct patients. the latter is based on molecular typing studies that have found indistinguishable strains of aspergillus terreus in patients with hematologic malignancies and in potted plants in the vicinity of the patients. [ ] [ ] [ ] the desired quality of air may be achieved without incurring the inconvenience or expense of laminar airflow. , to prevent inhalation of fungal spores during periods when construction, renovation, or other dust-generating activities that may be ongoing in and around the health care facility, it has been recommended that severely immunocompromised patients wear a high-efficiency respiratory protection device (eg, an n respirator) when they leave the pe. , , the use of masks or respirators by hsct patients when they are outside of the pe for prevention of environmental fungal infections in the absence of construction has not been evaluated. a pe does not include the use of barrier precautions beyond those indicated for standard precuations and transmission-based precautions. no published reports support the benefit of placing patients undergoing solid organ transplantation or other immunocompromised patients in a pe. these recommendations are designed to prevent transmission of infectious agents among patients and hcws in all settings where health care is delivered. as in other cdc/hicpac guidelines, each recommendation is categorized on the basis of existing scientific data, theoretical rationale, applicability, and, when possible, economic impact. the cdc/hicpac system for categorizing recommendations is as follows: category ia. strongly recommended for implementation and strongly supported by well-designed experimental, clinical, or epidemiologic studies. category ib. strongly recommended for implementation and supported by some experimental, clinical, or epidemiologic studies and a strong theoretical rationale. category ic. required for implementation, as mandated by federal and/or state regulation or standard. category ii. suggested for implementation and supported by suggestive clinical or epidemiologic studies or a theoretical rationale. no recommendation; unresolved issue. practices for which insufficient evidence or no consensus regarding efficacy exists. health care organization administrators should ensure the implementation of recommendations specified in this section. agents into the objectives of the organization's patient and occupational safety programs. assume that every person is potentially infected or colonized with an organism that could be transmitted in the health care setting and apply the following infection control practices during the delivery of health care. iv.a. . during the delivery of health care, avoid unnecessary touching of surfaces in close proximity to the patient to prevent both contamination of clean hands from environmental surfaces and transmission of pathogens from contaminated hands to surfaces. airborne precautions does not need to wear a mask or respirator during transport if the patient is wearing a mask and infectious skin lesions are covered. category ii v.d. . exposure management immunize or provide the appropriate immune globulin to susceptible persons as soon as possible after unprotected contact (ie, exposure) to a patient with measles, varicella, or smallpox: category ia d administer measles vaccine to exposed susceptible persons within hours after the exposure or administer immune globulin within days of the exposure event for high-risk persons in whom vaccine is contraindicated. , - d administer varicella vaccine to exposed susceptible persons within hours after the exposure or administer varicella immune globulin (vzig or an alternative product), when available, within hours for high-risk persons in whom vaccine is contraindicated (eg, immunocompromised patients, pregnant women, newborns whose mother's varicella onset was , days before or within hours after delivery). , - d administer smallpox vaccine to exposed susceptible persons within days after exposure. vi. protective environment (see table airborne infection isolation room (aiir). formerly known as a negative-pressure isolation room, an aiir is a single-occupancy patient care room used to isolate persons with a suspected or confirmed airborne infectious disease. environmental factors are controlled in aiirs to minimize the transmission of infectious agents that are usually transmitted from person to person by droplet nuclei associated with coughing or aerosolization of contaminated fluids. aiirs should provide negative pressure in the room (so that air flows under the door gap into the room), an air flow rate of to air changes per hour (ach) ( ach for existing structures, ach for new construction or renovation), and direct exhaust of air from the room to the outside of the building or recirculation of air through a highefficiency particulate air filter before returning to circulation. ( ambulatory care setting. a facility that provides health care to patients who do not remain overnight; examples include hospital-based outpatient clinics, non-hospital-based clinics and physician offices, urgent care centers, surgicenters, free-standing dialysis centers, public health clinics, imaging centers, ambulatory behavioral health and substance abuse clinics, physical therapy and rehabilitation centers, and dental practices. bioaerosol. an airborne dispersion of particles containing whole or parts of biological entities, including bacteria, viruses, dust mites, fungal hyphae, and fungal spores. such aerosols usually consist of a mixture of monodispersed and aggregate cells, spores, or viruses carried by other materials, such as respiratory secretions and/or inert particles. infectious bioaerosols (ie, those containing biological agents capable of causing an infectious disease) can be generated from human sources (eg, expulsion from the respiratory tract during coughing, sneezing, talking, singing, suctioning, or wound irrigation), wet environmental sources (eg, high-volume air consitioning and cooling tower water with legionella) or dry sources (eg, construction dust with spores produced by aspergillus spp). bioaerosols include large respiratory droplets and small droplet nuclei (cole ec. ajic ; : - ) . caregiver.. any person who is not an employee of an organization, is not paid, and provides or assists in providing health care to a patient (eg, family member, friend) and acquire technical training as needed based on the tasks that must be performed. cohorting. in the context of this guideline, this term applies to the practice of grouping patients infected or colonized with the same infectious agent together to confine their care to one area and prevent contact with susceptible patients (cohorting patients). during outbreaks, health care personnel may be assigned to a cohort of patients to further limit opportunities for transmission (cohorting staff). colonization. proliferation of microorganisms on or within body sites without detectable host immune response, cellular damage, or clinical expression. the presence of a microorganism within a host may occur with varying durations but may become a source of potential transmission. in many instances, colonization and carriage are synonymous. droplet nuclei. microscopic particles , mm in size that are the residue of evaporated droplets and are produced when a person coughs, sneezes, shouts, or sings. these particles can remain suspended in the air for prolonged periods and can be carried on normal air currents in a room or beyond, to adjacent spaces or areas receiving exhaust air. engineering controls. removal or isolation of a workplace hazard through technology. an airborne infection isolation room, a protective environment, engineered sharps injury prevention device, and a sharps container are examples of engineering controls. epidemiologically important pathogen. an infectious agent that has one or more of the following characteristics: ( ) readily transmissible, ( ) a proclivity toward causing outbreaks, ( ) possible association with a severe outcome, and ( ) difficult to treat. examples include acinetobacter spp, aspergillus spp, burkholderia cepacia, clostridium difficile, klebsiella or enterobacter spp, extended-spectrum beta-lactamaseproducing gram-negative bacilli, methicillin-resistant staphylococcus aureus, pseudomonas aeruginosa, vancomycin-resistant enterococci, vancomycin-resistant staphylococcus aureus, influenza virus, respiratory syncytial virus, rotavirus, severe acute respiratory syndrome coronavirus, noroviruses, and the hemorrhagic fever viruses. hand hygiene. a general term that applies to any one of the following: ( ) handwashing with plain (nonantimicrobial) soap and water, ( ) antiseptic handwashing (soap containing antiseptic agents and water), ( ) antiseptic handrub (waterless antiseptic product, most often alcohol-based, rubbed on all surfaces of hands), or ( ) surgical hand antisepsis (antiseptic handwash or antiseptic handrub performed preoperatively by surgical personnel to eliminate transient hand flora and reduce resident hand flora). health care-associated infection (hai). an infection that develops in a patient who is cared for in any setting where health care is delivered (eg, acute care hospital, chronic care facility, ambulatory clinic, dialysis center, surgicenter, home) and is related to receiving health care (ie, was not incubating or present at the time health care was provided). in ambulatory and home settings, hai refers to any infection that is associated with a medical or surgical intervention. because the geographic location of infection acquisition is often uncertain, the preferred term is considered to be health care-associated rather than health care-acquired. healthcare epidemiologist. a person whose primary training is medical (md, do) and/or masters-or doctorate-level epidemiology who has received advanced training in health care epidemiology. typically these professionals direct or provide consultation to an infection control program in a hospital, long-term care facility, or health care delivery system (also see infection control professional). health care personnel, health care worker (hcw). any paid or unpaid person who works in a health care setting (eg, any person who has professional or technical training in a health care-related field and provides patient care in a health care setting or any person who provides services that support the delivery of health care such as dietary, housekeeping, engineering, maintenance personnel). hematopoietic stem cell transplantation (hsct). any transplantation of blood-or bone marrow-derived hematopoietic stem cells, regardless of donor type (eg, allogeneic or autologous) or cell source (eg, bone marrow, peripheral blood, or placental/umbilical cord blood), associated with periods of severe immunosuppression that vary with the source of the cells, the intensity of chemotherapy required, and the presence of graft versus host disease (mmwr ; : rr- ). high-efficiency particulate air (hepa) filter. an air filter that removes . . % of particles . . mm (the most penetrating particle size) at a specified flow rate of air. hepa filters may be integrated into the central air handling systems, installed at the point of use above the ceiling of a room, or used as portable units (mmwr ; : rr- ). home care. a wide range of medical, nursing, rehabilitation, hospice, and social services delivered to patients in their place of residence (eg, private residence, senior living center, assisted living facility). home health care services include care provided by home health aides and skilled nurses, respiratory therapists, dieticians, physicians, chaplains, and volunteers; provision of durable medical equipment; home infusion therapy; and physical, speech, and occupational therapy. immunocompromised patient. a patient whose immune mechanisms are deficient because of a congenital or acquired immunologic disorder (eg, human immunodeficiency virus infection, congenital immune deficiency syndromes), chronic diseases such as diabetes mellitus, cancer, emphysema, or cardiac failure, intensive care unit care, malnutrition, and immunosuppressive therapy of another disease process [eg, radiation, cytotoxic chemotherapy, anti-graft rejection medication, corticosteroids, monoclonal antibodies directed against a specific component of the immune system]). the type of infections for which an immunocompromised patient has increased susceptibility is determined by the severity of immunosuppression and the specific component(s) of the immune system that is affected. patients undergoing allogeneic hematopoietic stem cell transplantation and those with chronic graft versus host disease are considered the most vulnerable to health care-associated infections. immunocompromised states also make it more difficult to diagnose certain infections (eg, tuberculosis) and are associated with more severe clinical disease states than persons with the same infection and a normal immune system. infection. the transmission of microorganisms into a host after evading or overcoming defense mechanisms, resulting in the organism's proliferation and invasion within host tissue(s). host responses to infection may include clinical symptoms or may be subclinical, with manifestations of disease mediated by direct organisms pathogenesis and/or a function of cell-mediated or antibody responses that result in the destruction of host tissues. infection control and prevention professional (icp). a person whose primary training is in either nursing, medical technology, microbiology, or epidemiology and who has acquired specialized training in infection control. responsibilities may include collection, analysis, and feedback of infection data and trends to health care providers; consultation on infection risk assessment, prevention, and control strategies; performance of education and training activities; implementation of evidence-based infection control practices or those mandated by regulatory and licensing agencies; application of epidemiologic principles to improve patient outcomes; participation in planning renovation and construction projects (eg, to ensure appropriate containment of construction dust); evaluation of new products or procedures on patient outcomes; oversight of employee health services related to infection prevention; implementation of preparedness plans; communication within the health care setting, with local and state health departments, and with the community at large concerning infection control issues; and participation in research. certification in infection control is available through the certification board of infection control and epidemiology. infection control and prevention program. a multidisciplinary program that includes a group of activities to ensure that recommended practices for the prevention of health care-associated infections are implemented and followed by health care workers, making the health care setting safe from infection for patients and health care personnel. the joint commission on accreditation of healthcare organizations requires the following components of an infection control program for accreditation: ( ) surveillance: monitoring patients and health care personnel for acquisition of infection and/or colonization; ( ) investigation: identification and analysis of infection problems or undesirable trends; ( ) prevention: implementation of measures to prevent transmission of infectious agents and to reduce risks for device-and procedure-related infections; ( ) control: evaluation and management of outbreaks; and ( ) reporting: provision of information to external agencies as required by state and federal laws and regulations (see http://www.jcaho.org). the infection control program staff has the ultimate authority to determine infection control policies for a health care organization with the approval of the organization's governing body. long-term care facility (ltcf). a residential or outpatient facility designed to meet the biopsychosocial needs of persons with sustained self-care deficits. these include skilled nursing facilities, chronic disease hospitals, nursing homes, foster and group homes, institutions for the developmentally disabled, residential care facilities, assisted living facilities, retirement homes, adult day health care facilities, rehabilitation centers, and long-term psychiatric hospitals. mask. a term that applies collectively to items used to cover the nose and mouth and includes both procedure masks and surgical masks (see http://www.fda. gov/cdrh/ode/guidance/ .html# ). multidrug-resistant organism (mdro). in general, a bacterium (excluding mycobacterium tuberculosis) that is resistant to or more classes of antimicrobial agents and usually is resistant to all but or commercially available antimicrobial agents (eg, methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing or intrinsically resistant gram-negative bacilli). nosocomial infection. derived from greek words, ''nosos'' (disease) and ''komeion'' (to take care of), refers to any infection that develops during or as a result of an admission to an acute care facility (hospital) and was not incubating at the time of admission. personal protective equipment (ppe). a variety of barriers used alone or in combination to protect mucous membranes, skin, and clothing from contact with infectious agents. ppe includes gloves, masks, respirators, goggles, face shields, and gowns. procedure mask. a covering for the nose and mouth that is intended for use in general patient care situations. these masks generally attach to the face with ear loops rather than ties or elastic. unlike surgical masks, procedure masks are not regulated by the food and drug administration. protective environment. a specialized patient care area, usually in a hospital, with a positive air flow relative to the corridor (ie, air flows from the room to the outside adjacent space). the combination of high-efficiency particulate air filtration, high numbers (. ) of air changes per hour, and minimal leakage of air into the room creates an environment that can safely accommodate patients with a severely compromised immune system (eg, those who have received allogeneic hemopoietic stem cell transplantation) and decrease the risk of exposure to spores produced by environmental fungi. other components include use of scrubbable surfaces instead of materials such as upholstery or carpeting, cleaning to prevent dust accumulation, and prohibition of fresh flowers or potted plants. quasi-experimental study. a study undertaken to evaluate interventions but do not use randomization as part of the study design. these studies are also referred to as nonrandomized, pre-/postintervention study designs. these studies aim to demonstrate causality between an intervention and an outcome but cannot achieve the level of confidence concerning an attributable benefit obtained through a randomized controlled trial. in hospitals and public health settings, randomized control trials often cannot be implemented due to ethical, practical, and urgency reasons; therefore, quasi-experimental design studies are commonly used. however, even if an intervention appears to be effective statistically, the question can be raised as to the possibility of alternative explanations for the result. such a study design is used when it is not logistically feasible or ethically possible to conduct a randomized controlled trial, (eg, during outbreaks). within the classification of quasi-experimental study designs, there is a hierarchy of design features that may contribute to validity of results (harris et al. cid : : . residential care setting. a facility in which people live, minimal medical care is delivered, and the psychosocial needs of the residents are provided for. respirator. a personal protective device worn by health care personnel over the nose and mouth to protect them from acquiring airborne infectious diseases due to inhalation of infectious airborne particles , mm in size. these include infectious droplet nuclei from patients with mycobacterium tuberculosis, variola virus [smallpox], or severe acute respiratory syndrome and dust particles that contain infectious particles, such as spores of environmental fungi (eg, aspergillus spp). the centers for disease control and prevention's national institute for occupational safety and health (niosh) certifies respirators used in health care settings (see http://www.cdc.gov/niosh/topics/respirators/). the n disposable particulate, air-purifying respirator is the type used most commonly by health care personnel. other respirators used include n- and n- particulate respirators, powered air-purifying respirators with high-efficiency filters, and nonpowered fullfacepiece elastomeric negative pressure respirators. a listing of niosh-approved respirators can be found at http://www.cdc.gov/niosh/npptl/respirators/disp_part/ particlist.html. respirators must be used in conjunction with a complete respiratory protection program, as required by the occupational safety and health administration, which includes fit testing, training, proper selection of respirators, medical clearance, and respirator maintenance. respiratory hygiene/cough etiquette. a combination of measures designed to minimize the transmission of respiratory pathogens through droplet or airborne routes in health care settings. the components of respiratory hygiene/cough etiquette are ( ) covering the mouth and nose during coughing and sneezing, ( ) using tissues to contain respiratory secretions with prompt disposal into a no-touch receptacle, ( ) offering a surgical mask to persons who are coughing to decrease contamination of the surrounding environment, and ( ) turning the head away from others and maintaining spatial separation (ideally . feet) when coughing. these measures are targeted to all patients with symptoms of respiratory infection and their accompanying family members or friends beginning at the point of initial encounter with a health care setting (eg, reception/triage in emergency departments, ambulatory clinics, health care provider offices). (srinivasin a iche ; : ; http://www.cdc.gov/flu/ professionals/infectioncontrol/resphygiene.htm). safety culture. shared perceptions of workers and management regarding the level of safety in the work environment. a hospital safety climate includes the following organizational components: ( ) senior management support for safety programs, ( ) absence of workplace barriers to safe work practices, ( ) cleanliness and orderliness of the worksite, ( ) minimal conflict and good communication among staff members, ( ) frequent safety-related feedback/training by supervisors, and ( ) availability of ppe and engineering controls. source control. the process of containing an infectious agent either at the portal of exit from the body or within a confined space. the term is applied most frequently to containment of infectious agents transmitted by the respiratory route but could apply to other routes of transmission, (eg, a draining wound, vesicular or bullous skin lesions). respiratory hygiene/cough etiquette that encourages individuals to ''cover your cough'' and/or wear a mask is a source control measure. the use of enclosing devices for local exhaust ventilation (eg, booths for sputum induction or administration of aerosolized medication) is another example of source control. standard precautions. a group of infection prevention practices that apply to all patients, regardless of suspected or confirmed diagnosis or presumed infection status. standard precautions represents a combination and expansion of universal precautions and body substance isolation. standard precautions are based on the principle that all blood, body fluids, secretions, excretions except sweat, nonintact skin, and mucous membranes may contain transmissible infectious agents. standard precautions include hand hygiene and, depending on the anticipated exposure, use of gloves, gown, mask, eye protection, or face shield. in addition, equipment or items in the patient environment likely to have been contaminated with infectious fluids must be handled in a manner to prevent transmission of infectious agents (eg, wear gloves for handling, contain heavily soiled equipment, properly clean and disinfect or sterilize reusable equipment before use on another patient). surgical mask. a device worn over the mouth and nose by operating room personnel during surgical procedures to protect both surgical patients and operating room personnel from transfer of microorganisms and body fluids. surgical masks also are used to protect health care personnel from contact with large infectious droplets (. mm in size). according to draft guidance issued by the food and drug administration on may , , surgical masks are evaluated using standardized testing procedures for fluid resistance, bacterial filtration efficiency, differential pressure (air exchange), and flammability to mitigate the risks to health associated with the use of surgical masks. these specifications apply to any masks that are labeled surgical, laser, isolation, or dental or medical procedure (http://www.fda.gov/cdrh/ode/guidance/ .html# ). surgical masks do not protect against inhalation of small particles or droplet nuclei and should not be confused with particulate respirators that are recommended for protection against selected airborne infectious agents (eg, mycobacterium tuberculosis). other species s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. giardia lamblia s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. noroviruses s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. persons who clean areas heavily contaminated with feces or vomitus may benefit from wearing masks, because virus can be aerosolized from these body substances; , , ensure consistent environmental cleaning and disinfection with focus on restrooms even when apparently unsoiled. , hypochlorite solutions may be required when there is continued transmission. [ ] [ ] [ ] alcohol is less active, but there is no evidence that alcohol antiseptic handrubs are not effective for hand decontamination. cohorting of affected patients to separate airs paces and toilet facilities may help interrupt transmission during outbreaks. rotavirus c di ensure consistent environmental cleaning and disinfection and frequent removal of soiled diapers. prolonged shedding may occur in both immunocompetent and immunocompromised children and the elderly. also for asymptomatic, exposed infants delivered vaginally or by c-section and if mother has active infection and membranes have been ruptured for more than to hours until infant surface cultures obtained at to hours of age negative after hours of incubation. susceptible hcws should not enter room if immune caregivers are available; no recommendation for face protection of immune hcws; no recommendation for type of protection (ie, surgical mask or respirator) for susceptible hcws. in an immunocompromised host with varicella pneumonia, prolong the duration of precautions for duration of illness. postexposure prophylaxis: provide postexposure vaccine as soon as possible but within hours; for susceptible exposed persons for whom vaccine is contraindicated (immunocompromised persons, pregnant women, newborns whose mother's varicella onset is # days before delivery or within hours after delivery) provide vzig, when available, within hours; if unavailable, use ivig. provide airborne precautions for exposed susceptible persons and exclude exposed susceptible health care workers beginning days after first exposure until days after last exposure or if received vzig, regardless of postexposure vaccination. variola (see smallpox) vibrio parahaemolyticus (see gastroenteritis) vincent's angina (trench mouth) s viral hemorrhagic fevers due to lassa, ebola, marburg, crimean-congo fever viruses s, d, c di single-patient room preferred. emphasize: use of sharps safety devices and safe work practices, hand hygiene; barrier protection against blood and body fluids on entry into room (single gloves and fluid-resistant or impermeable gown, face/eye protection with masks, goggles or face shields), and appropriate waste handling. use n or higher-level respirator when performing aerosol-generating procedures. largest viral load in final stages of illness when hemorrhage may occur; additional ppe, including double gloves, leg and shoe coverings may be used, especially in resource-limited settings where options for cleaning and laundry are limited. notify public health officials immediately if ebola is suspected. , , , also see table *type of precautions: a, airborne precautions; c, contact; d, droplet; s, standard; when a, c, and d are specified, also use s. y duration of precautions: cn, until off antimicrobial treatment and culture-negative; di, duration of illness (with wound lesions, di means until wounds stop draining); de, until environment completely decontaminated; u, until time 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hygiene compliance in an inpatient rehabilitation unit patient-education handbook learning styles and teaching strategies: enhancing the patient education experience handwashing: the semmelweis lesson forgotten? handwashing: simple, but effective elimination of methicillin-resistant staphylococcus aureus from a neonatal intensive care unit after hand washing with triclosan use of . % triclosan (bacti-stat) to eradicate an outbreak of methicillinresistant staphylococcus aureus in a neonatal nursery epidemiology and control of vancomycin-resistant enterococci in a regional neonatal intensive care unit hand hygiene and patient care: pursuing the semmelweis legacy a comparison of hand-washing techniques to remove escherichia coli and caliciviruses under natural or artificial fingernails impact of a -minute scrub on the microbial flora found on artificial, polished, or natural fingernails of operating room personnel bacterial carriage by artificial versus natural nails pathogenic organisms associated with artificial fingernails worn by healthcare workers postoperative serratia marcescens wound infections traced to an out-of-hospital source a prolonged outbreak of pseudomonas aeruginosa in a neonatal intensive care unit: did staff fingernails play a role in disease transmission? candida osteomyelitis and diskitis after spinal surgery: an outbreak that implicates artificial nail use outbreak of extended spectrum beta-lactamase-producing klebsiella pneumoniae infection in a neonatal intensive care unit related to onychomycosis in a health care worker impact of ring wearing on hand contamination and comparison of hand hygiene agents in a hospital bacterial contamination of the hands of hospital staff during routine patient care effectiveness of gloves in the prevention of hand carriage of vancomycin-resistant enterococcus species by health care workers after patient care efficacy of gloves in reducing blood volumes transferred during simulated needlestick injury performance of latex and nonlatex medical examination gloves during simulated use latex allergy and gloving standards a review of natural-rubber latex allergy in health care workers barrier protection with examination gloves: double versus single leakage of latex and vinyl exam gloves in high-and low-risk clinical settings in-use barrier integrity of gloves: latex and nitrile superior to vinyl latex and vinyl examination gloves. quality control procedures and implications for health care workers integrity of vinyl and latex procedure gloves occupational exposure to bloodborne pathogens: final rule. cfr part : recommendations for preventing the spread of vancomycin resistance: recommendations of the hospital infection control practices advisory committee (hicpac) examination gloves as barriers to hand contamination in clinical practice removal of nosocomial pathogens from the contaminated glove: implications for glove reuse and handwashing a mrsa outbreak in an sicu during universal precautions: 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infected patients unusual nosocomial transmission of mycobacterium tuberculosis value of the face mask and other measures droplet infection and its prevention by the face mask eye splashes during invasive vascular procedures guidance for industry and fda staff: surgical masks. premarket notification [ (k)] submissions; guidance for industry and fda national institute for occupational health and safety. eye protection for infection control the use of eye-nose goggles to control nosocomial respiratory syncytial virus infection respiratory syncytial virus (rsv) infection rate in personnel caring for children with rsv infections: routine isolation procedure versus routine procedure supplemented by use of masks and goggles rsv outbreak in a paediatric intensive care unit occupational safety and health administration. respiratory protection respiratory protection as a function of respirator fitting characteristics and fit-test accuracy respiratory protection against mycobacterium 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infection nosocomial pertussis in healthcare workers from a pediatric emergency unit in france an outbreak of multidrugresistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome update: severe acute respiratory syndrome transmission of mycobacterium tuberculosis to and from children and adolescents infection control in cystic fibrosis: practical recommendations for the hospital, clinic, and social settings probable secondary infections in households of sars patients in hong kong contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? disinfection and sterilization in health care facilities: what clinicians need to know outbreak of multidrug-resistant enterococcus faecium with transferable vanb class vancomycin resistance pseudomonas aeruginosa outbreak in a haematology-oncology unit associated with contaminated surface cleaning equipment role of environmental cleaning in controlling an outbreak of acinetobacter baumannii on a neurosurgical intensive care unit pseudomonas aeruginosa wound infection associated with a nursing home's whirlpool bath use of audit tools to evaluate the efficacy of cleaning systems in hospitals survival and vehicular spread of human rotaviruses: possible relation to seasonality of outbreaks acquisition of clostridium difficile from the hospital environment environmental control to reduce transmission of clostridium difficile transmission of rotavirus and other enteric pathogens in the home rotavirus infections in infection control reference service comparison of the effect of detergent versus hypochlorite cleaning on environmental contamination and incidence of clostridium difficile infection healthcare infection control practices advisory committee committee (hicpac) persistent acinetobacter baumannii? look inside your medical equipment computer equipment used in patient care within a multihospital system: recommendations for cleaning and disinfection computer keyboards as reservoirs for acinetobacter baumannii in a burn hospital computer keyboards and faucet handles as reservoirs of nosocomial pathogens in the intensive care unit reduction in vancomycin-resistant enterococcus and clostridium difficile infections following change to tympanic thermometers a randomized crossover study of disposable thermometers for prevention of clostridium difficile and other nosocomial infections bacterial surface contamination of patients' linen: isolation precautions versus standard care isolating and double-bagging laundry: is it really necessary? available from tracking perinatal infection: is it safe to launder your scrubs at home? mcn home-versus hospital-laundered scrubs: a pilot study double-bagging of items from isolation rooms is unnecessary as an infection control measure: a comparative study of surface contamination with single-and double-bagging recommended antimicrobial agents for the treatment and postexposure prophylaxis 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preventing transmission of infection with human t-lymphotropic virus type iii/lymphadenopathy-associated virus in the workplace severe acute respiratory syndrome measures for the prevention and control of respiratory infections in military camps efficiency of surgical masks in use in hospital wards: report to the control of infection subcommittee wearing masks in a pediatric hospital: developing practical guidelines handwashing and respiratory illness among young adults in military training effect of infection control measures on the frequency of upper respiratory infection in child care: a randomized, controlled trial the effect of hand hygiene on illness rate among students in university residence halls what is the evidence for a causal link between hygiene and infections? american association of nurse anesthesists. reuse of needles and syringes by healthcare providers put patients at risk. available from www.aana.com/news.aspx?ucnavmenu_tsmenutargetid & ucnavmenu_tsmenutargettype 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are effective in reducing bacterial contamination caused by dispersal from the upper airway guidelines for the prevention of intravascular catheter-related infections anxiety and depression in hospitalized patients in resistant organism isolation methicillin-resistant staphylococcus aureus: psychological impact of hospitalization and isolation in an older adult population the experience of respiratory isolation for hiv-infected persons with tuberculosis the experience of infectious patients in isolation contact isolation in surgical patients: a barrier to care? adverse effects of contact isolation do physicians examine patients in contact isolation less frequently? a brief report management of multidrug-resistant organisms in healthcare settings respiratory syncytial viral infection in children with compromised immune function nosocomial outbreak of parvovirus b infection in a renal transplant unit prolonged shedding of multidrug-resistant influenza a virus in an immunocompromised patient adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity chronic enteric virus infection in two t-cell-immunodeficient children prolonged shedding of rotavirus in a geriatric inpatient staphylococcus aureus nasal colonization in a nursing home: eradication with mupirocin attempts to eradicate methicillin-resistant staphylococcus aureus from a long-term-care facility with the use of mupirocin ointment natural history of colonization with vancomycin-resistant enterococcus faecium high rate of false-negative results of the rectal swab culture method in detection of gastrointestinal colonization with vancomycin-resistant enterococci recurrence of vancomycin-resistant enterococcus stool colonization during antibiotic therapy duration of colonization by methicillin-resistant staphylococcus aureus after hospital discharge and risk factors for prolonged carriage persistent contamination of fabric-covered furniture by vancomycin-resistant enterococci: implications for upholstery selection in hospitals aspergillosis due to carpet contamination flower vases in hospitals as reservoirs of pathogens nosocomial aspergillosis: environmental microbiology, hospital epidemiology, diagnosis and treatment aspergillus terreus infections in haematological malignancies: molecular epidemiology suggests association with in-hospital plants masking of neutropenic patients on transport from hospital rooms is associated with a decrease in nosocomial aspergillosis during construction the infection control nurse in us hospitals, - : characteristics of the position and its occupant are there regional variations in the diagnosis, surveillance, and control of methicillin-resistant staphylococcus aureus? results of a survey of work duties of infection control professionals (icps): are new guidelines needed for the staffing of infection control (ic) programs? critical care unit bedside design and furnishing: impact on nosocomial infections the ability of hospital ventilation systems to filter aspergillus and other fungi following a building implosion increased catheter-related bloodstream infection rates after the introduction of a new mechanical valve intravenous access port joint commision on accreditation of healthcare organizations. comprehensive accredication manual for hospitals: the official handbook new technology for detecting multidrugresistant pathogens in the clinical microbiology laboratory employee health and infection control nosocomial outbreak of pseudomonas cepacia associated with contamination of reusable electronic ventilator temperature probes ventilator temperature sensors: an unusual source of pseudomonas cepacia in nosocomial infection centers for disease control and prevention. bronchoscopy-related infections and pseudoinfections decontaminated single-use devices: an oxymoron that may be placing patients at risk for cross-contamination centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) control of influenza a on a bone marrow transplant unit impact of implementing a method of feedback and accountability related to contact precautions compliance evaluation of the contribution of isolation precautions in prevention and control of multi-resistant bacteria in a teaching hospital the text as an orientation tool surveillance for nosocomial infections monitoring hospitalacquired infections to promote patient safety controlling methicillin-resistant staphylococcus aureus: a feedback approach using annotated statistical process control charts spread of stenotrophomonas maltophilia colonization in a pediatric intensive care unit detected by monitoring tracheal bacterial carriage and molecular typing the impact of bedside behavior on catheter-related bacteremia in the intensive care unit epidemiology of invasive group a streptococcus disease in the united states regional dissemination and control of epidemic methicillin-resistant staphylococcus aureus. manitoba chapter of chica-canada emergence of community-associated methicillin-resistant staphylococcus aureus usa genotype as a major cause of health care-associated blood stream infections survival of hepatitis b virus after drying and storage for one week failure of bland soap handwash to prevent hand transfer of patient bacteria to urethral catheters skin tolerance and effectiveness of two hand decontamination procedures in everyday hospital use replace hand washing with use of a waterless alcohol hand rub? transmission of staphylococci between newborns: importance of the hands to personnel hands as route of transmission for klebsiella species effectiveness of hand washing and disinfection methods in removing transient bacteria after patient nursing extensive environmental contamination associated with patients with loose stools and mrsa colonization of the gastrointestinal tract efficacy of selected hand hygiene agents used to remove bacillus atrophaeus (a surrogate of bacillus anthracis) from contaminated hands banning artificial nails from health care settings prospective, controlled study of vinyl glove use to interrupt clostridium difficile nosocomial transmission latex glove penetration by pathogens: a review of the literature pcr-based method for detecting viral penetration of medical exam gloves association of contaminated gloves with transmission of acinetobacter calcoaceticus var. anitratus in an intensive care unit epidemiology and prevention of pediatric viral respiratory infections in health-care institutions nosocomial transmission of rotavirus from patients admitted with diarrhea safety and cleaning of medical materials and devices surface fixation of dried blood by glutaraldehyde and peracetic acid role of environmental contamination in the transmission of vancomycin-resistant enterococci disinfection of hospital rooms contaminated with vancomycin-resistant enterococcus faecium role of environmental contamination as a risk factor for acquisition of vancomycin-resistant enterococci in patients treated in a medical intensive care unit federal insecticide, fungicide, and rodenticidal act usc et seq is methicillin-resistant staphylococcus aureus (mrsa) contamination of ward-based computer terminals a surrogate marker for nosocomial mrsa transmission and handwashing compliance? transfer of bacteria from fabrics to hands and other fabrics: development and application of a quantitative method using staphylococcus aureus as a model evaluation of bedmaking-related airborne and surface methicillin-resistant staphylococcus aureus contamination bacterial contamination on the surface of hospital linen chutes designing linen chutes to reduce spread of infectious organisms iatrogenic contamination of multidose vials in simulated use: a reassessment of current patient injection technique a large outbreak of hepatitis b virus infections associated with frequent injections at a physician's office a large nosocomial outbreak of hepatitis c and hepatitis b among patients receiving pain remediation treatments patient-to-patient transmission of hepatitis c virus through the use of multidose vials during general anesthesia an outbreak of hepatitis c virus infections among outpatients at a hematology/oncology clinic streptococcal meningitis following myelogram procedures a prospective study to determine whether cover gowns in addition to gloves decrease nosocomial transmission of vancomycin-resistant enterococci in an intensive care unit parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome parainfluenza virus infection after stem cell transplant: relevance to outcome of rapid diagnosis and ribavirin treatment serial observations of chronic rotavirus infection in an immunodeficient child an outbreak of 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listeria moncytogenes cross-contamination in a nursery neonatal listeriosis due to cross-infection confirmed by isoenzyme typing and dna fingerprinting outbreak of neonatal listeriosis associated with mineral oil neonatal cross-infection with listeria monocytogenes nosocomial malaria and saline flush plasmodium falciparum malaria transmitted in hospital through heparin locks nosocomial malaria from contamination of a multidose heparin container with blood hospital-acquired malaria transmitted by contaminated gloves clustering of necrotizing enterocolitis: interruption by infection-control measures how contagious is necrotizing enterocolitis? an outbreak of rotavirus-associated neonatal necrotizing enterocolitis increased risk of illness among nursery staff caring for neonates with necrotizing enterocolitis outbreak of adenovirus pneumonia among adult residents and staff of a chronic care psychiatric facility nosocomial adenovirus infection: molecular epidemiology of an outbreak a recent outbreak of adenovirus type infection in a chronic inpatient facility for the severely handicapped an outbreak of multidrugresistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents human-to-human transmission of rabies virus by corneal transplant human rabies prevention, united states, : recommendations of the advisory committee on immunization practices (acip) rhinovirus and the lower respiratory tract concurrent outbreaks of rhinovirus and respiratory syncytial virus in an intensive care nursery: epidemiology and associated risk factors rhinovirus infection associated with serious lower respiratory illness in patients with bronchopulmonary dysplasia nosocomial ringworm in a neonatal intensive care unit: a nurse and her cat nosocomial transmission of trichophyton tonsurans tinea corporis in a rehabilitation hospital molecular epidemiology of staphylococcal scalded skin syndrome in premature infants an outbreak of fatal nosocomial infections due to group a streptococcus on a medical ward an outbreak of group a streptococcal infection among health care workers clusters of invasive group a streptococcal infections in family, hospital, and nursing home settings isolation techniques for use in hospitals us government printing office rethinking the role of isolation practices in the prevention of nosocomial infections the authors and hicpac gratefully acknowledge dr larry strausbaugh for his many contributions and valued guidance in the preparation of this guideline. the mode(s) and risk of transmission for each specific disease agent listed in this appendix were reviewed. principle sources consulted for the development of disease-specific recommendations for the appendix included infectious disease manuals and textbooks. , , the published literature was searched for evidence of person-to-person transmission in health care and non-health care settings with a focus on reported outbreaks that would assist in developing recommendations for all settings where health care is delivered. the following criteria were used to assign transmission-based precautions categories: d a transmission-based precautions category was assigned if there was strong evidence for person-to-person transmission via droplet, contact, or airborne routes in health care or non-health care settings and/or if patient factors (eg, diapered infants, diarrhea, draining wounds) increased the risk of transmission. d transmission-based precautions category assignments reflect the predominant mode(s) of transmission. d if there was no evidence for person-to-person transmission by droplet, contact or airborne routes, then standard precautions were assigned. d if there was a low risk for person-to-person transmission and no evidence of health care-associated transmission, then standard precautions were assigned. d standard precautions were assigned for bloodborne pathogens (eg, hbv, hcv, hiv) in accordance with cdc recommendations for universal precautions issued in . subsequent experience has confirmed the efficacy of standard precautions to prevent exposure to infected blood and body fluid. , , additional information relevant to use of precautions was added in the comments column to assist the caregiver in decision-making. citations were added as needed to support a change in or provide additional evidence for recommendations for a specific disease and for new infectious agents (eg, sars-cov, avian influenza) that have been added to appendix a. the reader may refer to more detailed discussion concerning modes of transmission and emerging pathogens in the background text and for mdro control in the mdro guideline. key: cord- -rndtf authors: brosseau, lisa m; rosen, jonathan; harrison, robert title: selecting controls for minimizing sars-cov- aerosol transmission in workplaces and conserving respiratory protective equipment supplies date: - - journal: ann work expo health doi: . /annweh/wxaa sha: doc_id: cord_uid: rndtf with growing evidence of inhalation of small infectious particles as an important mode of transmission for sars-cov- , workplace risk assessments should focus on eliminating or minimizing such exposures by applying the hierarchy of controls. we adapt a control banding model for aerosol-transmissible infectious disease pandemic planning to encourage the use of source and pathway controls before receptor controls (personal protective equipment). built on the recognition that aerosol-transmissible organisms are likely to exhibit a dose–response function, such that higher exposures result from longer contact times or higher air concentrations, this control banding model offers a systematic method for identifying a set of source and pathway controls that could eliminate or reduce the need for receptor controls. we describe several examples for workers at high risk of exposure in essential or return to work categories. the goal of using control banding for such workers is to develop effective infection and disease prevention programs and conserve personal protective equipment. there is growing evidence of inhalation of small infectious particles as an important mode of sars-cov- transmission (asadi et al., ; bahl et al., ; heinzerling et al., ; meselson, ; morawska and cao, ; wang and du, ) . the covid- pandemic is likely to continue for some time and workplace outbreaks continue to occur, even in locations where community transmission has decreased. from that perspective, occupational hygienists have an obligation annals of work exposures and health, , - doi: . /annweh/wxaa original article to consider hazardous sars-cov- aerosols in workplace risk assessments and to encourage employers to utilize well-studied and proven source and pathway control strategies for minimizing aerosol exposures. we describe a qualitative risk assessment and control selection approach for protecting employees from aerosol-transmissible diseases, such as covid- , and illustrate how such an approach could be used during an ongoing pandemic where essential workers must remain at work and, eventually, most employees must physically return to work. we currently lack the tools to adequately measure employee exposures to inhalable concentrations of infectious organisms. in contrast to airborne chemical exposures, well-validated air sampling and analytic methods for infectious aerosols are not readily available. while many chemical agents have well-established occupational exposure limits, the infectious dose by inhalation exposure route has not been identified for most organisms. these factors limit our ability to quantify exposure levels and correlate exposures with health outcomes. without these key risk assessment ingredients, our ability to evaluate occupational risk and effectiveness of controls for infectious aerosols is limited. when the levels of exposure to a workplace hazard are not known or difficult to measure and workplace exposure limits are not available or difficult to construct, occupational health and safety professionals may resort to qualitative tools, such as control banding, for determining which occupations, tasks, and industries present the highest risk for workers (zalk and heussen, ) . these tools can also be useful in determining the best control options for lowering exposures. absent a pandemic, human-generated infectious respiratory diseases generally occur in workplaces such as healthcare, childcare, protective services, laboratory research, and funeral services among those with the highest exposure likelihood (haagsma et al., ) . during a pandemic, when community spread of infection is high, exposures could occur in all workplaces where there is close contact or prolonged time spent with clients, patients, customers, or coworkers. control banding is a qualitative method for determining the degree of risk for occupations and job tasks. first used in the pharmaceutical industry, control banding allowed identification of appropriate measures for reducing exposures to novel materials for which few toxicologic or epidemiologic data were available (zalk and nelson, ) . more recent applications have been in regulatory settings where quantitative exposure limits are unavailable (russell et al., ) and for novel substances such as nanomaterials where toxicologic data are rare (paik et al., ) . identifying the correct 'hazard band' requires knowledge of toxicity (of the substance or a close relative), the nature of exposure (physical state, route of entry, likely airborne concentration), and duration of exposure. lacking quantitative data, descriptive values may be used, for example: low, medium, high concentration; and short, medium, long duration of exposure. the hazard band is accompanied by a required or recommended set of control measures, commensurate with the level of risk and informed by the hierarchy of controls. sietsema et al. ( ) proposed a control banding method for aerosol-transmissible diseases, such as covid- , for two reasons: (i) to identify those jobs at highest risk and (ii) encourage the use of source and pathway controls before resorting to personal protective equipment (ppe), for the ultimate goal of conserving ppe for those in the highest risk categories. their approach uses four risk groups to represent toxicity, ranging from r for agents not associated with disease in healthy adults to r being agents likely to cause serious or lethal human disease. this is combined with an exposure band that derives from three categories of duration and three exposure likelihood categories. once these factors are determined one of three control bands (a, b, or c) will result for a workplace or set of job tasks, which then informs the selection of appropriate source, pathway, and receptor controls. the goals of this work are to describe how the sietsema et al. ( ) identify organism toxicity lacking a dose-response function for infectious diseases, sietsema et al. ( ) propose instead applying the national institutes of health (nih) risk groups developed for laboratory biosafety applications (national institutes of health, ). the risk group represents the 'toxicity' of an organism, in its broadest sense, categorizing an organism by its ability to cause disease in humans, the seriousness of that disease, and the availability of preventive (e.g. vaccines) or therapeutic (e.g. drugs) interventions. the nih office of science policy has determined that sars-cov- is a risk group organism (national institutes of health, ). for a fraction of the population, covid- is a very serious and sometimes lethal disease for which there are not yet any well-established preventive or therapeutic interventions. an employee's exposure to a hazardous airborne chemical depends on two variables: (i) air concentration and (ii) length of time in contact with that air concentration. for sars-cov- , we lack routine and widely available quantitative methods for sampling airborne infectious organism concentrations and there is uncertainty regarding the infectious dose required to cause disease. we know that infectious dose varies for different organisms, suggesting there is some exposure level (concentration of infectious aerosols) required to elicit infection and disease (ward et al., ) . as well, infectious dose varies with route of entry. while aerosol exposure has only rarely been explored experimentally, the infectious dose by aerosol inhalation for some respiratory organisms such as influenza is lower than that by other exposure routes (lednicky et al., ; belser et al., ) . setting aside these issues, as well as disease outcome differences by age, gender, race, comorbidities, and other host factors, we assume that sars-cov- requires a certain 'dose' of infectious particles to achieve respiratory infection that results in viral shedding. we assume that a cumulative dose resulting from a short exposure to a high aerosol concentration will be just as likely to lead to infection as one resulting from a longer exposure (such as an -h work shift) to a lower concentration. thus, exposure is a function of (i) the likelihood that the job involves person-to-person interactions with potentially infected people and (ii) the total duration of time-over the course of a day or shift-such personto-person interactions occur. likelihood of exposure ranges from 'unlikely' to 'possible' to 'likely', while duration ranges from to , to , or > h. the latter was predicated on an -h shift; this variable could also be expressed as percent or another metric appropriate to the job. these variables are used to determine the level of exposure as shown in table , where e represents low likelihood of exposure, e possible exposure of short or medium duration and e likely exposure of medium or long duration. the exposure level (e , e , or e ) is then combined with the organism risk ranking to identify the relevant 'control band' for a job (table ) . each control band requires implementing preventive measures starting with the source, then pathway and finally receptor (fig. ) . control band a, which is reserved for risk group organisms or unlikely exposures for risk group and organisms, requires the use of only source and then, if necessary, pathway controls. receptor controls (personal protective equipment) are not appropriate for jobs that fall into this band. jobs falling into control band b are those where exposure is unlikely but the toxicity (risk group) is severe, or where exposures are possible or likely and risk is moderate (risk groups and ). controls for band b should be focused primarily on sources and then, if necessary, pathways; receptor controls might be necessary but only as a final resort. only in control band c, reserved for possible and likely exposures and higher risk groups, should receptor controls, such as respiratory protection, be considered, after source and pathway controls, of course (table ) . the most effective and appropriate set of control strategies must start with those that prevent or minimize release of a hazard, in this case, the infectious aerosol, at the source, i.e. an infected individual. during a pandemic source control is complicated by the fact that every person present in a workplace-coworkers, customers, visitors, etc.-could be a source of infectious aerosols. aerosolization or reaerosolization of infectious particles from a surface could also be a source. with sars-cov- we are furthered hampered by evidence that a great deal of transmission appears to occur pre-and asymptomatically. thus, we must assume that every person in a workplace is a potential source. the prevalence of pre-and asymptomatic transmission ranges from to % in some settings (kimball, ; mizumoto et al., ; wei et al., ) . daily duration (number of potential exposure hours per h workday) examples of source controls might be: • eliminate all in-person interactions by requiring customers to conduct their business via phone, internet, or some remote method, in combination with delivery or drive-by pickup. this type of control has been applied successfully in some locations to the purchase of perishable goods such as groceries and prepared food. • conduct employee health screenings prior to arrival at work, to identify and exclude those potentially infected (note: this may not identify pre-or asymptomatic individuals). • clean surfaces and eliminate activities that may contribute to reaerosolization of deposited infectious particles (e.g. the use of compressed air to remove particulates or clean surfaces). • eliminate person-to-person meetings by requiring remote communication methods. • adjust work schedules to limit the number of people present in a workplace at any one time. • reorganize congregate locations (lunchrooms, bathrooms, conference rooms, entrances, and exits) to limit the number of people. if source controls do not adequately prevent or minimize employee exposure to infectious aerosols, by limiting likelihood or duration, then pathway controls must be implemented in tandem. such controls eliminate or limit the movement of infectious aerosols from the source (infected person) to the receptor (employee). pathway controls might include: • place barriers that interrupt the flow of infectious aerosols between the source and receptors. • increase the amount of dilution air (air changes per hour). • designate 'hot' and 'cold' or 'clean' and 'dirty' zones that separate infectious sources from receptors. • incorporate distancing into settings with many sources, keeping in mind that particles from coughs and sneezes can easily travel beyond ft. • utilize local exhaust ventilation to capture infectious aerosols at the source. • use high-efficiency portable air cleaners to remove particles near the source and improve air mixing throughout a space. pathway controls are, in general, more difficult to design and implement correctly. for example, increasing the number of air changes per hour (dilution ventilation) can lower the overall concentration of particles in a space, but may not be effective at lowering particle concentrations near the source. industrial hygienists do not typically employ dilution ventilation for the control of hazardous aerosols from industrial processes, because it will not protect workers near the source. relying on dilution ventilation allows distribution of particles throughout a space and could increase everyone's exposure to particles in comparison to local exhaust ventilation, which removes particles at the source. in a space with a good directional airflow design using multiple intakes and exhausts that ensure good air mixing, dilution ventilation may be effective. enclosing the process or partial enclosure in combination with local exhaust can be much more effective at protecting everyone, including those nearest the source. where people are the source of infectious aerosols, a better option might be the use of portable air cleaners equipped with high-efficiency filters, if the construction of local exhaust ventilation is not immediately possible. care must be taken in the placement of such devices, as they may change the movement of air throughout the space and raise exposure for some workers while lowering it for others. the use of barriers to enclose or divide the space into smaller compartments may be useful in tandem with portable air cleaners, but this also requires careful design to ensure proper mixing within the enclosed spaces. it would be appropriate to consult with an expert in the design of such control systems. many occupational hygienists have the expertise and equipment to assess aerosol generation and movement throughout a workplace. some have expertise in the design and evaluation of local exhaust ventilation systems and similar types of source controls. as this discussion illustrates, multiple pathway controls may be necessary to ensure that either exposure likelihood and/or exposure duration are minimized. only as a last resort, receptor controls might be necessary. in the case of a pandemic, when receptor controls such as personal protective equipment are in short supply, every effort must be made to reserve supplies for workers whose exposures place them in control band c and for workers whose jobs cannot easily be controlled using only source and pathway methods. for workplaces where employees may be at higher risk of serious illness for reason of age or preexisting conditions, it may be appropriate to adjust the control band or select additional control measures. because healthcare workers are one group at highest risk, while source and pathway controls should be utilized and can be effective at lower exposures, personal protective equipment, such as respirators, will also be necessary. as examples, we consider control banding for some essential workers who have been working throughout the pandemic as well as those whose risks may be high after stay-at-home orders are lifted. controls for front-line healthcare workers are not addressed here; these can be found in us and european guidelines (centers for disease control and prevention, b; european centre for disease prevention and control, ). transportation is considered an essential industry, important for ensuring that other essential workers can make it to work. bus and subway drivers have constant contact with others, work directly with the public, provide personal assistance to customers, spend most of their worktime in an enclosed vehicle and are often in moderate proximity to people (within an arm's length) (department of labor, ). exposure likelihood is very high (l ) for these jobs, given the potential for the presence of many pre-or asymptomatic infected people. transit workers spend most of their working hours in contact with people (d ), thus their exposure level is e (table ) . when combined with a risk rank of r for covid- these workers fall into control band c. in workplace settings we would deploy local exhaust ventilation to capture and remove hazardous aerosols at the source, but this is an impractical solution for transit passengers. in the longer term, retrofitting buses or subway cars with localized ventilation that captures and transports aerosols away from each seat might be a possibility. limiting the number of passengers would limit the airborne concentration of infectious particles, thus lowering the overall aerosol concentration to which passengers and drivers are exposed. this may be a workable solution but would require running more buses or cars more frequently, which would require more drivers, a perhaps infeasible solution in the short term. pathway controls, i.e. preventing the flow of particles from passengers into the driver's breathing zone, may be more amenable to inexpensive and effective solutions. one example might be the isolation of the driver in a separately ventilated enclosure. there are well-established methods that would allow communication between passengers and drivers. automated solutions for collecting money and dispensing tickets are already available on most mass transit, thus limiting requirements for direct contact. if well-designed, complete enclosure would eliminate exposure to passengergenerated aerosols and lower exposure to l (unlikely), thus decreasing exposure to e and moving this job into control band a. in this case, there is no need to resort to further pathway controls or to require the use of any personal protective equipment. the work of stock or warehouse clerks involves daily and frequent face-to-face discussions, constant contact with others, working with groups or teams and spending about half of the time in moderately close proximity to other people (at arm's length) (department of labor, a). for the purposes of this example, we assume that exposure to infected coworkers is likely (l ), given the annals of work exposures and health, , vol. xx, no. xx amount of pre-and asymptomatic transmission reported for covid- . we assume a daily duration of exposure from to h (d ) since some of the shift probably involves working on one's own. this yields an exposure of e and results in control band c. source control (limiting emission of aerosols from infected coworkers) may be possible by symptom screening, with follow-up testing (if available), to limit the number of infected people present. screening may not identify people who are pre-or asymptomatic, however. isolation of workers using enclosures is not feasible for this type of work, where employees must move throughout a large space. limiting worker interactions by employing remote methods for communication, such as phones or walkie-talkies, may be a better solution. requiring employees to work at a designated distance from each other, limiting the number of employees in a particular area or on a shift, changing the manner in which work and teams are organized are all methods that will interrupt the pathway from potential sources to receptors. increasing the rate of dilution ventilation (air changes per hour) or adding local exhaust ventilation may not have much impact on the generation of aerosol from sources (coworkers) continually moving throughout a space, although dilution ventilation could lower the overall concentration in smaller spaces, such as lunchrooms, toilets, and other communal locations. if source and pathway controls do not lower the likelihood of exposure from l to l it may be necessary to decrease the duration of exposure from d by decreasing the length of shifts, for example. as before, lowering the likelihood of exposure to l decreases the overall exposure to e and moves this job into control band a. no receptor controls (personal protective equipment) should be necessary. one of the first physicians who called attention to and subsequently died of exposure to sars-cov- in china was an ophthalmologist (parrish et al., ) . a large majority of the work of an ophthalmologist requires close, face-to-face interactions with patients while conducting eye examinations and surgeries, administering medications, or performing diagnostic or clinical tests. the work involves assisting and caring for others, communicating, and working directly with patients and coworkers and coordinating the work and activities of others. the work involves close contact with others % of the time, daily face-to-face discussion, dealing with members of the public, working with a group or team, potential exposure to diseases or infections, and being in very close proximity to people (nearly touching) more than % of the time (department of labor, b). given the number of pre-and asymptomatic people with covid- and the lack of definitive tests, every patient could be infected. it may be possible to require patients to wear a surgical mask, which may limit some number of larger droplets but will not prevent the emission of small particles during breathing or talking. it may also be possible limit the number of patient interactions by tele-medicine. it may be possible to devise some form of local exhaust ventilation, such as a slot hood, near the patient, which would remove particles from the immediate vicinity. such controls can be very efficient at removing hazardous aerosols if well designed and operated at appropriate flow rates. a barrier between the patient and physician might also be appropriate, in combination with local exhaust ventilation, to direct particles away from the breathing zone. this would require some careful design by someone with expertise in ventilation design along with evaluation, using tracer particles or gas for example, to ensure that the configuration is effective at removing particles from the space. using a barrier without ventilation may prevent movement of particles into the breathing zone of the physician during the exam but would not limit particle distribution and buildup in the room. small particles can remain suspended in air for considerable time; after several infected patients, the concentration in a small exam room may exceed the infectious dose. if source controls do not adequately lower aerosol concentrations, then pathway controls may also be necessary. one option might include the addition of a standalone high efficiency particulate air-filtered air cleaner to the room, which would enhance air movement throughout the space while also collecting infectious particles. the sizing and placement of such an air cleaner are important, to ensure adequate and effective air movement and cleaning. again, consultation with an expert may be necessary. it is important that air and particles are drawn away from the source and not through the breathing zone of the physician. another option would be to build a ventilated enclosure around the patient or the physician. the former would require a filter to clean exhausted air, while the latter would require filtering the air before it enters the enclosure. either of these options may be infeasible if they interfere with conduct of an eye examination. given the close contact nature of an eye examination, it may be necessary to employ personal protective equipment such as respiratory protection in addition to the source and pathway controls described. an n filtering facepiece respirator would be the minimum level of respiratory protection recommended for this exposure. eye protection would also be recommended, as recent data suggest that eye exposure may lead to upper respiratory infection (li et al., a) . goggles or a face shield in combination with a filtering facepiece respirator or a respirator with full-face protection would be appropriate, although these may interfere with communication and conduct of an eye exam. this workplace does not easily lend itself to source and pathway controls, but there may be some innovative approaches we have not considered. resorting to receptor controls (respiratory protection) without at least making some effort to design enclosures or deploy ventilation would not be appropriate. respirators are uncomfortable to wear for long periods of time, interfere with communication and may make it difficult to conduct eye examinations and tests. police patrol officers may engage in a wide range of activities, including responding to emergencies, maintaining order, protecting people and property, enforcing laws, providing aid, testifying in court, conducting patrols, directing traffic, issuing citations, etc. they work directly with the public, resolve conflicts and negotiate with people, communicate with supervisors, peers, and subordinates, assist and care for others, train and teach others, develop and build teams, coach and develop others. they are involved in daily face-to-face discussions, constant contact with others, deal with external customers, deal with unpleasant or angry people, work in groups or teams and spend at least half of their time very close (nearly touching) to people (department of labor, c). police officers have many interactions with many different members of the public and coworkers, any of whom could be infected and pre-or asymptomatic. exposure is likely (l ) and duration is either d ( - h) or d (> h). either duration yields an exposure level of e . combined with a risk level organism, this job falls in control band c. given the unpredictable nature of an officer's interactions with coworkers and the public, source controls may not be feasible. for people being held in the back seat of a patrol car, it should be possible to prevent aerosol movement to the front seat area using a barrier. such barriers may not be possible in other workplace locations, however. the best options may be limiting the amount of time spent in close contact with people and limiting the number of contacts. it may be necessary to reconfigure the job to reduce the time spent doing close-contact tasks. changing the configuration of desks, increasing or changing ventilation, adding more circulation and in-room filtration, utilizing more remote methods for communicating with the public are all possible methods for reducing contact time and number of contacts. pathway controls would appear to be more feasible and effective than source controls for this type of job. in some cases where contact time and number of contacts cannot be reduced, personal protective equipment (a fit-tested respirator) may be necessary. the most credible information about transmission of sars-cov- is that it occurs person-to-person (centers for disease control and prevention, a). transmission in group settings appears to be highly likely, such as families, meetings, religious and social gatherings, and funerals (james et al., ; li et al., b; pung et al., ; tong et al., ) . some of the risk factors for workers appear to be (dyal, ; gan et al., ; kinner et al., ; koh, ; koh and goh, ; lan et al., ; mcmichael et al., ; moriarty, ; payne et al., ; semple and cherrie, ; tobolowsky et al., ) : . contact with many people-either members of the public (e.g. healthcare workers, emergency responders, transportation workers, grocery store workers, correctional and immigration officers, retail salespeople, taxi drivers, casino workers, and cruise ships) or coworkers (warehouses, meatpacking plants, healthcare settings, and naval ships). . close prolonged face-to-face interactions with people-either members of the public (ophthalmology, dentistry, elder care, emergency, and acute patient care) or coworkers (police, emergency responders, retail workers, casino workers, manufacturing workers, and construction workers). . contact with suspected or confirmed infected people such as in healthcare settings and covid- testing sites, where infection risks are higher for those with more contacts, more close contacts, more prolonged contacts, or contact during symptoms or aerosolgenerating procedures. baker et al. ( ) , using data from the o*net database to identify occupations likely to be exposed to diseases or infections, found that % of healthcare support and healthcare practitioners and technical staff reported exposure at least once a week and greater than % reported exposure at least once a month. exposure at least once a month were reported by % of protective or personal care services workers, - % of workers in community and social services and education, training and libraries, and - % of building and grounds, cleaning and maintenance, and office and administrative support workers. obviously, these data represent non-pandemic circumstances; the likelihood for exposure during a pandemic will be considerably higher for these and other occupations, especially during accelerating phases of the community infection. in wuhan china, from the beginning of the pandemic in december to february , a total of healthcare workers were infected, . % of all cases. the attack rate in healthcare workers ( per people) was almost . times greater than for the general population ( per ). from january to february the attack rate among healthcare workers reached per . a report from the chinese center for disease control and prevention evaluated cases throughout china through february ; . % ( ) were healthcare workers; % were severe cases and deaths resulted (wu and mcgoogan, ) . lan et al. ( ) conducted an observational study of confirmed covid- cases reported in hong kong, japan, singapore, taiwan, thailand, and vietnam government investigation reports during the first days after the first local transmission case was reported. of local transmission, % ( ) were considered possibly work-related, with the highest frequency of cases in healthcare workers ( %), drivers and transport workers ( %), services and sales workers ( %), cleaning and domestic workers ( %), and public safety workers ( %). high-risk occupations included car, taxi, and van drivers, retail salespeople, domestic housekeepers, religious personnel, construction laborers, and tour guides. early in the transmission period the workers most likely to be infected were retail salespeople, driver, construction laborers, religious professionals, tour guides, and receptionists. later in the transmission period, healthcare workers, drivers, housekeepers, police, and religious personnel were at highest risk of infection. these data support the cases selected to illustrate jobs where sars-cov- exposures are likely to be high and control banding could be useful for identifying source and pathway controls. we were unable to find any workplace guidelines that adequately accounted for the role of aerosol transmission, i.e. the inhalation of aerosols near a source. many are overly focused on contact transmission, a relatively unimportant mode of transmission, and assume that droplet transmission by propulsion into the mouth, eyes, or nose from symptomatic individuals coughing or sneezing is the only particle-related transmission mode. guidance from the european agency for safety and health at work ( ) notes the importance of applying the hierarchy of controls, starting with eliminating the risk followed by minimizing worker exposure and finally personal protective equipment. recommendations for minimizing exposure rely on reducing physical contacts, increasing distance, using barriers, and reducing close contact time. there is no discussion of ventilation, but there is recognition that the placement of barriers could introduce new hazards. while recognizing that inhalation of infectious particles may be a possible route of exposure, the united states occupational safety and health administration (osha) covid- recommendations describe only very general categories of controls within the hierarchy, but do not address their applicability or feasibility in the context of risk level. limited attention is paid to methods for limiting aerosol transmission other than a brief mention of ventilation and somewhat greater attention to respirators, despite the latter being at the bottom of the control hierarchy. osha's four risk levels describe exposure in the context of contact with known or suspected sources of covid- and frequency of such contacts, but do not recognize the importance of contact time, airborne concentration, or proximity (department of labor, d). the control banding approach described here, which focuses on the likelihood and duration of exposure, is more useful for estimating risks. thinking about the control hierarchy in the context of source, pathway, and receptor controls offers a more nuanced approach for considering interventions, when every worker is a potential source. the focus on combining multiple source and pathway controls ensures that most workers will not be required to wear personal protective equipment. this model, as is true for similar models used for other hazardous exposures where occupational exposure limits are not available, has not undergone rigorous validation. while this is an important weakness, a model built on the well-established principles of dose-response and a hierarchy that emphasizes source and pathway controls are the core principles of occupational hygiene decision-making, which often requires a combination of well-informed science and professional judgment. no funding was provided for this project. the coronavirus pandemic and aerosols: does covid- transmit via expiratory particles? airborne or droplet precautions for health workers treating coronavirus disease ? estimating the burden of united states workers exposed to infection or disease: a key factor in containing risk of covid- infection comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza a viruses department of labor. ( a) o*net online summary report for: - . -stock clerks-stockroom, warehouse, or storage yard covid- among workers in meat and poultry processing facilities- states infection prevention and control for covid- in healthcare settings-third update preventing intra-hospital infection and transmission of covid- in healthcare workers infectious disease risks associated with occupational exposure: a systematic review of the literature transmission of covid- to health care personnel during exposures to a hospitalized patient high covid- attack rate among attendees at events at a church-arkansas asymptomatic and presymptomatic sars-cov- infections in residents of a long-term care skilled nursing facility-king county prisons and custodial settings are part of a comprehensive response to covid- occupational risks for covid- infection occupational health responses to covid- : what lessons can we learn from sars? work-related covid- transmission in six asian countries/areas: a follow-up study ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus a/ vietnam/ / (h n ) novel coronavirus disease (covid- ): the importance of recognising possible early ocular manifestation and using protective eyewear asymptomatic and human-tohuman transmission of sars-cov- in a -family cluster public health-seattle and king county, evergreenhealth, and cdc covid- investigation team. 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( ) investigation of three clusters of covid- in singapore: implications for surveillance and response measures an introduction to a uk scheme to help small firms control health risks from chemicals covid- : protecting worker health. ann work expo health a control banding framework for protecting the us workforce from aerosol transmissible infectious disease outbreaks with high public health consequences covid- outbreak among three affiliated homeless service sites potential presymptomatic transmission of sars-cov- covid- may transmit through aerosol minimum infective dose of animal viruses presymptomatic transmission of sars-cov- -singapore characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention banding the world together; the global growth of control banding and qualitative occupational risk management history and evolution of control banding: a review the authors declare no conflict of interest relating to the material presented in this article. its contents, including any opinions and/or conclusions expressed, are solely those of the authors. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -ak pq authors: nan title: th european congress of intensive care medicine athens - greece, october – , abstracts date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: ak pq nan objectives: evaluate the levels of tnf, il- and pai-i in different moments of the ards and the possible relationships among them. methods: septic patients with ards were studied. also significant differences for: tnf, pai-i and il- in septic patients and both evaluations of ards with control gropup; pai- between septics and nd evaluation in ards, and between the ist and nd evaluation in ards; il- between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il- , with clinical signs, are suggestive of infection; ) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; ) due to its own kynetics, il- takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution ( % hes, mean molecular weight (mw) , daltons, degree of substitution (ds) . ) or human albumin % (ha) for volume therapy for days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule- [selam -i] , intercellular adhesion molecule- [sicam -i] , vascular cell adhesion molecule- [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from • to • during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from • to , • sgmp- increased significatly only in the ha-group ( • to • only pao /fio was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the month period , patients were hospitalized on our intensive care unit. central venous catheters were examined for microbiological culture. specimens appeared to be possitive ( %). patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was / ( %). the incidence of sepsis due to the presence of all central venous lines was / ( %). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n= ), escherichia colt (n= ), others (n= ). in the epic study the percentage for sepsis on the icu was . % for the netherlands and . % for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than , ] ] ). results: five patients ( , %) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( . + . and . + . , respectively, p< , , mann-whitney u test). no patient with a saps greater than survived. mortality among the patients with saps from to was , %o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=- , , p= . ). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than , failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of neutropenic(wbc < , / )patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in from patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within minutes up to , hours after administration of antipyretics. three patients received , g of metamisol and one , g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level ( . + . ~ accompanied'by hypotension. but in - hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required + ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by other methods used in icu patients. methods: we prospectively studied adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution "d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and % limits of agreement (l of a = d_+ sd . td-fick - . - . to . fick-echo . - . to . there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n ; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il- , il- , il- , tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from . % to . % in patients with abscessed pneumonia and lung abscesses and from % to % if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il- . intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in % and mortality was decreased from % to %. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of ) rbct ; ) number of units transfused; and ) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from to and excluded patients who died in the first days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first days after onset of sepsis. overall -day mortality was %. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco (pico ) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico > mmhg. pentaspan| ( ml) was infused in rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico were performed before and repeated miff and hr after pentaspun| infusion. we calculated the pico -arterial pco' difference (pico -paco ) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was : : mmhg and lactate . : : . mmol/l. pentaspan| increased ci by % (p< . ) but did not change pico ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between and hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion , nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: . moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem : / . . moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med ; : - ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita mg/kg (tb), or varied doses of ec, followed hours later by bolus mg/kg iv lps. -day survival is shown below: group: s tb ec( . gmikgi ec( .sgm/kg) ec(i. gm/kg) alive (n) t ~ total (n) s s "signiflcant;y different from all other groups, p< . s / rats given lps followed hours later by ec ( . gm/kg) also died. additional rats were treated with s (n= ) or gm/kg ec (n= ) followed by mg/kg lps, then sacrificed at hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl ~/mm ), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, - scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = hz impedance (z; {dyn.sec.cm " }); zl = first harmonic z; zc = characteristic z; z ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < . between fio . and . , fio . and fio . &no - . _+ . - . _+ . # - . + . m - . + . * - . + . * - . + . * - . _+ . * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa ( + rnmhg versus + mmhg; p < . ). hypoxia increased (ppa-ppao) at all levels of q by an average of mmi-ig in minipigs and mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: ) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. ) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august to january , thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from to mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from . mm hg + . to . _+ . s.e.m., p= . , systemic vascular resistance ( + to + dyne.sec/ cm s, p=. ), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output ( . _+ . to . _+ . i/min p=. ) and increased pulmonary artery pressure ( . _+ . to . _+ . mm hg; p=. ) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of patients survived for more than days, patients died of cancer complications (all patients had maintained blood pressure for h on nma) and patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included mongrel dogs ( • pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of mg/kg of e. coli endotoxin, the dogs were randomized to receive a ml/kg infusion in rain of a hypertonic ( mosm]l) solution iucludiug either a mixture of glucose % with u insulin and meq kcl/l (glk-group ) or hydroxyethyl starch . % in naci . % (hes-group ). in each dog, a . % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and miu, rain, rain, and nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table ) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. , for group , for group ) were drawn and immediately analysed at ~ using the abl radiometer for po , pco and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the ps at ph= . , pco = mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps "in vivo" (i.e. the ps at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in patients aged from to with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of nm(mmi) & nm (mm ), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic ) and also some clinical and biochemical findings on the , , day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from . ~ . to . +. on the -th day after the operation. true decrease of the level mm from . ~. to . +. un & optimal density and increase of distribution index from . to . are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from . +. g/i to i . +. g/i on the -th day after the operation simultaneously with the decrease of cic from . ~ to . ~ . (p . ) were founded. some stages of the investigation true increase of lymphocytes from . + . % to . + . % was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=. -. in p<. ) was discovered and it also showed positive dynamics of the course of the disease. a year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin (il ) and (il ), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n= ), ards (n= ) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a rain resting period (baseline), then min after dopamine ( mcg/kg/min) infusion, and finally rain after the end of dopamine infusion (recovery gmbf _+ (perfusion units) gmbf ~a% vs baseline) * p < . vs "baseline" and "recovery". conclusions : ) despite a slight increase in co (+ %), the dramatical increase in gmbf (+ %) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. ) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and , -dihydroneopterin (nh ) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n ( pm), nh ( ilm), lipopolysaccharide (lps, ~g/ml), and interferone-~/(ifn-~/, u/ml) for h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn- seem to have a more than additive effect nh did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in patients, with sepsis, with severe sepsis and with septic shock, as well as in healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in anesthetized rats, catheters were placed in the femoral vein and artery. rats were given an iv bolus of lipopolysaccharide (lps, mg/kg), at baseline (to). after h (t ), rats received at random an infusion of either can ( mg/kg/h; can group, n=l ) or an equivalent volume of . % naci ( cc/kg/h; nac group, n= ), giyen over h (t -t ). a third group (sham group, n= ) received . % nac in place of lps, and then was treated like the nac group. mean blood pressure (mbp), blood lactate and nitrates (no ) were measured each h. glucose, creatinine and asat were also measured in rats (n= in each group). the can _+ * + "t . + . "~ . +_ . "t + " + " *p< . can vs naci ?p< . vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no , confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), patients with non septic shock (nss), patients with bacterial infection without shock ( nf) and icu patients without shock and without infection (control) were compared for proct levels at dayl, , , , . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p= . ), infection ( . +_ vs _+ ,p= . ), shock ( _+ vs +.- ,p= . ), death at day ( _+ vs _+ ,p= . ). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day proct levels ( = . ) in patients with shock, dayl proct levels are correlated with saps, infection and death at day , but not with arterial lactate levels (p= . ), white blood calls (p= . ) or fever (p= . ). proct levels remain higher i~i septic shock patients at day , and ( figure) . i c edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin ( gg/kg). the blood flow to the mucosal and the muscular layer was measured in regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map ( . _+ . vs . -+ . , p< . ), and phi ( . !-_ . vs . _+ . , p< . ), with a constant co ( _+ vs _+ , p= . ) and qst, aa ( . _+ . vs . _+ . , p= . ). the results of regional pertusion are presented in the table. (flow in ml/rain g of tissue; mean _+ sem ; * p< . vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a % increase in flow. acute increase in wbc count (from a mean of lo.oo mm a to o /mm~), between the rd and the th day of therapy. there was a decline of the wbc count to an average of about . mm a after decreasing the daily dose of the medication to mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c complement between . to . g/i. normal values . to . g/i there was no change in c values. conclusions : an early increase in wbc count was observed ( rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin- receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, h, h, h, h, and h after the administration of mg/kg (n= ) or mg/kg (n= ) of i - ra or the corresponding placebo (n = ). / patients ( %) were treated with adrenergie agents and / ( %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l beck from to . results: within the observation period s. was isolated from the stool cultures of patients. in patients (g m, f, median age yrs) s. could be detected in blood cultures ( s. enteritidis, s. typhimurium). in addition, in of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least up to days, of the patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids ( ), immunosuppressive drugs ( ), chronic alcoholism ( ), malignancies ( ), none ( ). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) + + death (n) duration of shock (h) + + noradrenaline (rag/h) , _+ + temperature (~ , + , + pvr (dynxsecxcm - ) + + co (ljmin) , _+ , , + , lactate (mmol/l) + , , + interleukin- (pg/ml) _+ + interleukin- (pg/ml) , _+ , , + , tnf-alpha (pg/ml) , + , + neopterin (nmol/l) , + , + crp (rag/l) _+ +_ pro-ct (ng/ml) , + , , + there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within h did not. however, in late cardiogenic shock (> h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine ( . mg/kg base in h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from / to / , pts with sfm (who ) treated with iv quinine for less than h were included prospectively. blood samples were collected at o, , , , , and h viability was assessed by culturing parasitized red blood cells in the presence of h-hypoxanthine, and radioactivity was determined at h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/ )xlo /icso for quinine (nmo]/]) was called the parasiticida/ index. results: pts were included, • saps . -+ . . the initial parasitemia was t. + . %. complications of malaria were coma ( pts), shock ( pts), renal failure ( pts) and acute lung injury ( pts). all strains were sensitive to quinine (icso -- nmol/ ). in pts who were not given a loading dose, parasite viability increased by and %, with concomitantly low quinine levels ( and #mow] at h); pt died. in pts that received a loading dose (serum quinine at h = . -- . ~mol/]) a marked decrease of parasite viability (by +_ % at h) was shown. viability was inversely correlated with plasma quinine (r=. , p-.o ) and parasiticidal index (r=. , p-.o ). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in consecutive admission to a university hospital icu in - and the icu and hospital survival of the bacteremia patients. during these years patients had positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of . % (vs. , % in all icu patients) and six month survival of . % (vs. . % in all icu patients). the most common bacteria were enterobacteriaceae ( , %), staphylococcus aureus ( , %) , coagulase negative staphylococci ( . %), pseudomonas ( . %) and slieptococci ( . %). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from - to - , records of consecutivepts with septic shock (crit care med , : - ) admitted to the icu were reviewed retrospectively. results: among pts with septic shock admitted during the study period, had hiv infection- of whom had aids-(gr. i) and were hiv-negative (gr. ill. ten gr. ii pts ( %) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in % gr. i and % gr. ii pts in gr . i pts ( %) a multivariate analysis demonstrated that hiv infection and sap i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il- and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il- , the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied critically ill patients ( men, women; - years old) in three diferents groups. group i: patients without evidence of infection, group i : patients with sepsis and with septic shock (group iii). we measured plasma il-lo, tnf and il- levels in the first hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il- were higher in group iii (median, pg/ml; range, - pg/ml) than in group ii (median, pg/ml; range, - pg/ml; p <. ) and group i (median, pg/ml; range, - pg/ml; p <. ). median il- concentrations did not differ among patients who survived (median pg/ml; range, - pg/ml) and those who died during the overall follow-up period ( days) (median, ; range, - pg/ml); but patients who died in short-term (< hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, pg/ml; range, - pg/ml). in patients with bacteriemia ( %), levels of il- were higher (median, pg/ml; range, - pg/ml) than in those with negative blood culture (median, , pg/ml; range - . pg/ml; p< . ). there was a good correlation between plasma il-io concentration and levels of tnf (r= . ; p < . ) and il- (r= . ; p < . ). the correlation between levels of il- and the apache ii score was significant only in the septic shock group (r= . ; p <. ). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il- levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital - lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : patients (age : • years ; saps ii : • were prospectively investigated (septic shock : bone criteria). following parameters were collected during hours : systemic hemodynamic parameters (swan ganz catheter a h -ref computer -baxter lab.), calculated systemic oxygen transport (do ), oxygen consumption (vo ) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco (pco ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter , cold z computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp- ) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < . ) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam- concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il- , il- and increased expressior of adhession molecules (selectin e, p, icam- ) measure of icam- and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi = . ). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/ o g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx ( . b , difco) in saline at mg/kg or h. both interventions produced % reduction in map within min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:( )baseline, ( ) mid-point of map reduction; ( ) nadir of hypotension, ( )midpoint of map recovery, & ( ) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o o:oc ., objectives : evaluate the relationship between il- , a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il and ils in severe sepsis and septic shock. methods : twenty-eight icu patients ( m, f, mean age + y) were studied as soon as they developped a severe sepsis (n = ) or a septic shock episode (n= ) as defined by a conference consensus in ( ). tnf~, il , il s and il- plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il- plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < . ) between il- and tnf a (r= . ), il- and il~ (r = . ) and il- and il s (r = . ) as well as between il- and apache n score (r= . ). patients who died (n = ) had il- levels higher than patients who survived but this difference was not statistically significant ( pg/ml vs . pg/ml; p> . ). conclusions : during severe sepsis and sepsis shock, il- seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : ( ) crit care med ; : - . objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin ( . mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least mmhg and/or a decrease in the necessary vasoactive medication of at least % within hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than nmol/l. results: of patients were found to respond to steroid medication, did not. mean cortisol levels before and after corticotropin were • and • nmol/l in the responder group (rg) and • and • nmol/l in the non responder group (nrg). in the rg out of ( %) were found to have an adrenal insufficiency, in the nrg out of ( %). in the rg -weeks mortality was . % (l out of ), the overall mortality % ( out of ). mortality in the nrg was % ( out of ) (p < . ) and % ( out of ) (p < . ) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam- and s-tnt levels continuously starting at the beginning of sepsis. methods: patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within hrs following the beginning of sepsis. blood was drawn every hrs in the first ;~ hrs, after hrs, followed once per day for days. s-tnt, icam- , elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) . ( . - . ), . ( . - ), . ( . - . ) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. . ( . - . ) . patients had s-tnt levels > . pg/l. of these died, whereas only of patients died with s-tnt values < . pg/l (p= . ). all patients that died had elevated sjcam- levels ( ilg/l:cut-off ) whereas in the survivor group only % had elevated icam- levels (p= , ). conclusions: increased slcam- and s-tnt levels were found during early sepsis in the majority of patients, a high sicam- and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on critically ill postoperative septic patients (their multiple organ failure score was - /with icg monitor. in cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during - hours in every minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on patients using previously defined criteria. methods: normotensive patients with fever (temperature > . ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients ( %) had or developed sirs within the first three days, patients ( %) did not. sepsis was present in % at inclusion. in patients with sirs, % did not progress to sepsis or septic shock, % progressed to sepsis (mean interval . • . days), and patient (< %) directly progressed from sirs to septic shock. in patients with sepsis, % progressed to septic shock (mean interval . • . days). sepsis was preceded by sirs in %. septic shock was preceded by sepsis in % and by sirs in %. conclusions: % of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. patients with double pneumonia were subjected to the evaluation. sh in the concentration of mg/l in the volume of - m / hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence . - times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, ' , h ) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by % in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. , goris et al arch. surg. , kuaus et al ann. surg. colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il- with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for - hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, o ng/ml lps was required to increase cgmp levels, whereas the presence of % fbs shifted the lps concentration curve i times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (> ~ min) but not removal of small molecules (< , d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin -~ (il-i). % fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed > hours following admission to our icu, during a year period ( january through january ) were reviewed. patients (pts) were included, mean age , y (sd , ), saps , (sd , ), mac cabe ( and ) %, mortality directly due to sepsis %. pts had mrsa bacteremia and methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age ( , + , vs , + , ) , saps ( , + , vs , + , ), use of vancomycin ( % vs %), mechanical ventilation ( % vs %), number of days (d) before the drawing of the first positive blood culture (median d, range - d vs median d, range - d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) ( % vs % p< , ), central venous catheter infection due to staph.aureus ( , % vs % p< , ), but previous use of antibiotics was not significantly different ( , % vs %). the outcome of the bacteremic pts was not statistically different: saps at the first day of bacteremia ( , +_. , vs , + , ), severe sepsis and septic shock ( % vs %), persistence of the bacteremia ( % vs %), mortality directly due to bacteremia ( % vs %). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio ; : - ) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of dead cases had been analized. results: in cases ( , %) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in cases ( %) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in cases ( %) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of dead cases ( %) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs ( kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a minute intravenous infusion )f escherichia colt (serotype h , sigma, st. louis, mo) was begun ~t a rate of pg/kg. an infusion of either saline at . ml/kg/min (group ; n= ) or saline and dobutamine at a rate of pg/kg/min (group ii; n= ) vas begun mn after the end of the endotoxin infusion. tesults : to td t ~ fl w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: patients with sepsis (criteria according to accp/sccm, ) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output< ml/ h, cr> . mg/dl and bun> mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on , and hours. cytokines tnfa, i - and ii- were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and h solute mass transfer of tnf and i - were calculated (v h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included pts and group b pts with mean+sd age ( _+ vs _+ , ns) and apache scores( _+ vs -+ . , ns). the mean ta-+ sd was . + vs -+ , p< . . the mean_+se morbidity period of group b vs group c was _+ vs _+ . p< . . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was . and for i - was . . the solute mass tranfer was -fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, delivery (do ) and consumption (c ). objectives: the main purpose of this article is to determine the relationship betwee~ delivery an consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d and v . we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after hours. high levels of do and v may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january and december . specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of patient there were ( , ~) adults and ( , ( ~) children. almost two thirds of the patients ( - , ~) were males. the predominantly cause ( , ~) of children's burns was scalding b~y hot liquids and flame burns ~ , ~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa ( " in adult patients) and staphyloccocus epidermidis ( , % in children). in only five patients ( , ~ the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in , % of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was , ~, but the mortality rate caused of sepsis was low (i %) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do (vigilance ~ baxter), v (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t ) na infusion ( , to , hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp ) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il and il were determined every hours for days and on day after fulfilling the criteria of ss and severe sepsis. results : in pts, sepsis was caused by a gram (-) microorganism, in pts by a gram (+) and in pts no microorganism was identified. there were survivors ( %) (s) and non-survivors ( %) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il- were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl ( c ) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion >_ .i ]tg/kg/min with a map _< mmhg. cardiovascular support was limited to na _+ dobutamine (db), c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t = ); and at the end of treatment (t = ) with c . conclusions: c can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. c is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t ); vl, dobu and na. results: in patients with treated ss, tests were performed (vl n= ; dobu n= ; na n= method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion ~> . ~g/kg/min with a map _< mmhg. cardiovascular support was limited to na + dobutamine (db), c was administered for up to h at a fixed dose-rate of either i, . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at h from the start of treatment (t = ); and at the end of treatment (t - ) with c . conclusions: c is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. c is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc ( c ) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . [xg/kg/ min with a map _< mmirlg. cardiovascular support was limited to na + dobutamine. c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = ); at the end of treatment (t = ); and h after treatment (t = ) with c . results. -median values (* assessment made at h or when c discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to c . the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of c in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc ( cg ) given by continuous infusion for h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . ~tg/kg/min with a map _< mmhg. cardiovascular support was limited to na • dobutamine. c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. plasma was collected from each patient over a h period and analysed for c . pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/ -plasma elimination halflife). conclusion: the pharmacokinetics of c in patients with septic shock are dose-independent at infusion rates up to . mg/kg/h. at higher rates, clearance of c decreases without any marked change in volume of distribution. c metabolism may be partially saturable at dose-rates above . mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc ( c ) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd- mice ( - g) were instrumented under gaseous anaesthesia (isofluorane, %) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after h recovery, endotoxin administration (e. col• :b , - . mgkg - i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from + to + mmhg (n= , p< . ) at h, with a survival rate at h, h and h of %, % and % respectively. c administered as a h continuous infusion ( mgkg-th -t i.v., n= ), h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from + to + mmhg (n= ) at h, with an improved survival rate at h, h and h of %, % and % respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using c represents a novel and promising treatment for septic shock. cultures of e.coli ( , %) and candida( , %) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia ( , %cultures) and common e.coli( , %)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in . objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of months all surgical patients on icu were registrated prospectively, more than parameters for each of them were documented'daily in a paradox file. results (see table ): of patients fulfilled the criterion of a septic shock (r. bone, ) , of them died at the lth day, while the surviving group of patients stayed almost days at icu. obiectives: to compare the effects of and % pentastarch solutions to a human albumin solution on oxygen delivery (do ) in septic patients. methods: this stud}, included septic patients with fever (t > ~ tachycardia flqr > /rain), tachypnea (rr > /min) or mechanical ventilation, leukocytosis (wbc> /mm ) or leukopcnla (wbc< ()/mm ) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < mmhg. patients were randomized to receive ml of % albunun (n:i ), hydroxyethyl starch (hes -mw /d.s. . ) % (n: ) or t % (n=i ); patients were also treated with adrenergic agents. results cardiac index (c ) increased significantly only in % lies (table) hemoglobin (hb) decreased significantly at min in the same group. there was not significant change in oxygen delivery ( do ). baseline ci alb . :: . (l'min/m ) hes % . = . hes % . polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after days ( day = onset of septic shock ) in % of the patients pci was demonstrable. after days % of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. . pci develops early in the course of illness ( % after days ). . pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: ) can serial measurements of vapco and avph also reflect the onset of tissue hypoxia during endotoxemia? ) are whole body changes in vapco and avph associated with parallel changes in regional circulation? methods: in anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo ). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n= ), group (n-- ) received mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do . results: systemic do crit was higher ( . _+ . vs . + . ml/kg.min, p < . ) and critical oxygen extraction ratio (o ererit) was lower ( . +_ . vs . +_ . %, p< . ) in the endotoxie than in the control group. meslenteric and femoral do crit were higher in the endotoxic than in the control group ( . _+ . vs . _+ . mlll g tissue.min and . _+ . vs . _+ . ml/min, respectively, both p< . ). regional o ercrit were lower in we endotoxic than in the control group (mesenteric: . _+ . vs .i_+ . %, renal: . + . vs . -+ . % and femorah . -+ . vs . _+ . %, all p< . ). vapco and avph followed a similar pattern in both groups, increasing slightly before do crit was reached, but dramatically wh,en do fell below do crit. in the presence like in the absence of endotoxin, systemic and regional do crit calculated from vo , from vapco , or from avph were similar; do crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. ) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: anesthetized dogs received endotoxin ( mg/kg iv), followed min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo ) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n= ) maintained low arterial pressure and systemic vascular resistance. the dogs receiving a continuous infusion of , , ~ug/kg.min of sin- starting min following initial fluid resuscitation (each dose for h), had a higher cardiac index ( . -+ . vs . _+ . l/min.kg, p< . ) and lower systemic and pulmonary vascular resistance than the control group ( -+ vs -+ and -+ vs -+ dyne.sec/cm , respectively, both p< . ). arterial pressure and pulmonary artery pressure were not influenced. sin- significantly increased the mesenteric blood flow from -+ to + % of baseline levels and renal blood flow from -+ to -+ % (both p< . ) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo , oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin- increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv- g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) - il- , and soluble (s) e-selectin were measured in both tcv- and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv versus placebo (takeda chemical industries ltd., osaka, japan). tcv- was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock ( . mg/kg body weight intravenously over hrs, twice a day for one week). day mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining patients patients were randomised to receive tcv- and to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il- and il- , considered to reflect severity of disease, tended to be higher in tcv treated patients: . . vs . ng/ml for il- (p. ) and . vs . ng/ml for il- (p. ) in tcv- and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day i.e. the end of tcv treatment (mortality: out of in the tcv group and out of patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il- and plasma il- levels tended to decrease more rapidly in tcv treated patients compared to controls (p=. and p=. , for il- and /l- respectively) conclusions: tcv- is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv- may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged - y.) with verified septicemia had been included in this study. beginning from day oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days - . patients, f and m died @u= ring their stay at the icu, of them in less than h~urs: after admission. at the begin surviving patients showed olab values between - %, non-survivors between - %, com= pared to neopterin values of - m~ol/l in survivors and - ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in consecutive children on day after uncomplicated open heart surgery was compared to gtt in children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o, g/kg in a % aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within minutes in / . after closed heart surgery gtt was delayed (+- ) min vs. (+- ) compared to controls, but transit was achieved in all aptients within rain. mean dopamine dose was , mcg/kgmin after cpb and , meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge ', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only newborns, b.w. - g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were + , and aado , -+ , kpa. results. newborns ( %) died shortly alter" admission or during transport without possibity to further is newborns ( , %) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in newborns ( , %) and of them survived the mortality rate of group mentioned was %. conclusion. the fact that in the czech republic ( mil. population with natality rate of about . newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, - , ltb and thromboxane b release in whole blood: each of . ml hepariaised blood of healthy adult volunteers was iucubated lbr hears with eillier ill ug/ml lps, mg/ml b&,ine smfactant (alveulhct/themae), rag/nil artificial surfactant (exosurf/wellcome), og lps + i mg/ml bovine surfactant or i ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. ' e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of . ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr hours with eilher aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of - but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with . ~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b ) and the lipooxygenase (lencotrieae b ) pathways. conclusion: we cooclude: ) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. ) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. ) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope , tcpco or san leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo and lb content, capillary flow rate and oxygen uptake rate. aim of the study: ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. ) to investigate the possibility of obtainiug infonnations about the local nptake and the venous as well as arterial satnration. patients and methods:we investigaled non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope , tcpco and san were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded spectres within minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo vahms were sorted by a software package. on the assumption that the low values correspond to the tibo value of the veaons ends of the capillaries and the high values to those of the arterial ends we ) it is possible to gel infonnations about the local o -uptake and arterial saturation ia the peripheral tissue by photoslg'ctroscopy. ) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from( - )ws.continnous positive air way pressure was applied to six cases at peep level from ( - )cm h o through nasal pronge,(group i),the other cases were managed as routine,(group ii).blood gases, tcpo ,tcco ,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among ( . %)of cases,while ( . %)of cases need imv.the cases of group ii need imv among ( %)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel , the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all ( ) pediatric intensive care units (icus) in the netherlands ( tertairy and non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality aenee, standardized mortality rate). efficiency was determined by objective criteria (mortality risk > % or administration of at least icu-dependent therapy)'. consecutive admissions aged < years old were included. overall, observed and expected mortality were in good agreement (p > . ). between hospitals, crude mortality showed wide variations (mean . %, range - %). however, in each center, observed and expected mortality were similar (mean ratio . , range . - . ). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was %, however large variations between units were found (range: - %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the -bed neonatal intensive care unit of a university hospital material and method: a total of consecutive outbem newborns admitted to nlod from , . to ., . were enrolled to the study. babies who died or were discharged from the unit within hours of treatment were excluded from the study, mean birth weight was g (+/- r), mean gestatational age was weeks (+/- . wks), mean age at the admission was h (+/- hi. multiple (~_ j organ system failure occurred jn . % of babies at the admission./~tertal lactates were measure/at the admission, among - hour and - hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was . %, mean length of nicu stay for survivors was . days (+/- . day). we found high lactate levels at the admission in . % babies (~ . % with levels above . retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of years was recorded in patients ( . %), patients ( . %) reported a - year remission and patients ( . %) had a remission lasting less than months before they became pregnant. results: seven patients ( . %) developed medium attacks in the second half of pregnancy, four patients ( . %) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first - weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during - weeks. a significant portion of pregnant women with b.a. ( %) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight ( - gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about % of newborns weighing below g who are born before weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i -ml and -ml pump volume. the right sided pumps are % smaller for biventricular use. between february and may we implanted this assist system in children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in infant. patient n.l: kg, lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl mcg/kg/h, hypocapnia of mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at ppm, and the gas aoalyser for no and no (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/ of systemic was obtained with a dramalic improvement. no was continued at ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu days after. patient n. : . kg, monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was % wilh a pap at % of systemic. two hours of c wenlional therapy failed o improve ihc patient and no administration was slarled at ppm. so dramatically incrcased to %, but the pap dropped only to % of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at ppm in the following days. she was extubaled days after surgery and discharged from the icu days after. patient n. : kg, 'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after d;b's and disclmrged from the icu after days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case : severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after days ( hours) -the patient was extubated weeks later. case : tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis ( days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: ) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. ) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to near term neonates with severe persistent pphn, oxygenation index > and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the patients studied responded to the initial trial of no ( ppm x minutes), as defined by a greater than percent improvement in pad as well as a fall in the el to < . these patients were continued on ino therapy, with patterns of response emerging: pattern babies (n= ) continued to show a sustained response to ino and were successfully weaned from it within days -all survived. pattern babies (n= ) failed to sustain their response to ino over hours, as definded by a rise in the el > . six survived, five with ecmo. pattern babies (n= ) had a sustained dependence on ino for - weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern ) not only required ino for longer periods of time than did the sustained responders (pattern ), but also required significantly higher doses of ino we report on the air transport of paediatric intensive care patients. these transports fall into three categories: ) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = ). one patient was transferred on ind. mean transfer time . hours (se + . hrs). ) long distance international transport using chartered aircraft (n = ). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time . hours (se + . hrs) ) long distance international transport using commercial aircraft (n = ). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time hours (se _+ .fhrs, longest hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; ) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication ) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. ) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials ( hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_. children,asa i-if,median age years,range - who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( children,age - yrs,ketamine , mg/kg, diazepam - mg total dose ) or per os ( children,ketamine - mg/kg,diazepam , mg/kg or midazclam , - , mg/kg ) _resu_l_t_s. - minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted - minutes and allowed adequate needle emg and nerve conduction investigation. children recieved additional , - , vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was neonates admitted during the years to , with gestational age _< weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo ) < . ), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo before the administration of the st dose of surfeclant, delivery, apgar score at min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was . %. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: . , % c : . , . ). for every . on average decrease of a/apo before surfactant administration the risk of p-ivh in the neonates was . times higher ( % ci: . , . ). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: . , % ci: . , . ). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of ( boys, girls) accidental ingestions of foreign bodies in children (mean age : . years, range : months- years). no child had ingested more than foreign object. the majority of the ingested foreign bodies were : coins (n : ), toy parts (n : ), jewellery (n : ), batteries (n : ), "sharp" materials such as needles and pins (n : ), "large" amounts of food (n : ). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. / patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : hours, range min. - hours). chest and/or abdominal x-ray was performed as first-line investigation ( / objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in children. in / the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from . to years. methods: protocol n consisted of intravenous administration of diphtheria antitoxic serum, prednisolone ( mg/kg bw/day), plasmapheresis and supportive care. protocol n included the use of antitoxic serum against the background of high-dose dexasone ( - mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n consisted of children with malignant diphtheria, of them with severe malignant diphtheria (grade and ). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the children in the group died seven. the children of the second group were treated according to the protocol n . out of total of patients of this group. patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of children admitted to the trial survived, one child with malignant diphtheria of grade and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. - , l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a months period non-neonatal pediatric ards patients (age - months) in a "pre-ecmo"-situation (oi over h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia ( ), sepsis ( ), immunosuppression ( ), near drowning ( ), neurogenous ards ( ). a total of - mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. hrs. if there was no improvement of the oi by at least , further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of mg/kg in doses. patients with no benefit (oi remained unchanged or increased within the max. - hrs) were taken on ecmo. results: nine patients improved within hours after surfactant therapy: the oi decreased from a level of (mean, range - ) before our treatment to a level of (mean, range - ) thereafter. in patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within - days. the other patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient : in a boy with down's syndrome an avsd had been surgically corrected at month of age. at , yr of age a catheter examination revealed a pulmonary vascular resistance of % of systemic vascular resistance in room air and at an fin of . . prostacyclin ( . mcg/ml) was administered with a jet nebulizer at an fin of . . pvr declined to . systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion ( ng/kg rain) had to be stopped after minutes because of systemic arterial hypotension. patient : a month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing % oxygen via face mask pao was mmhg, arterial ph was . . systolic arterial pressure was mmhg, a rv-ra gradient of mmhg was measured by cw-doppler. while fio was maintained aerosolized prostacyclin was administered over minutes. rv-ra gradient was mmhg, systemic blood pressure mmhg, pao mmhg. two hours later nitric oxide ( ppm) was inhaled at an fio of ( , . rv-ra gradient declined from to mmhg, systemic systolic blood pressure remained stable at mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient the poor response to inhaled pgi compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years ( - ), infants weighing less than gr. admitted to our referral unit. thirty four of them ( %) survived, ( % of infants weighing - g and % of infants weighing - gr survived) for the years - - the survival of these infants was % and for the years - - , % (p< . ). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors ( ) to neonatal deaths ( ) shows: gestational age: . w ( ) to . w ( ) (s). birth weight: . g ( ) to . ( ) (s). apgar score: , ( ) to . ( ) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants . ",,( ) to % ( ) (s) use of surfactant: % ( ) to % ( ). bpd observed in % of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in % of the mothers. in conclusion: ) a great improvement in the survival rate observed in these infants the last years in our unit. ) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated healthy preterms less then weeks postnatal age (gestational age +_ . wks; x + sd, weight +_ g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair . . + twall . ). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress ( rain). ii) insulation increased by a cap ( min), iii) min after its removal, iiii) increased operant temperature by . + . ~ ( min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded ( ) or diagnosticated an upper pouch fistula ( ). in patients who suspected "h" fistula ( ) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible - french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients ( primary anastomosis, resections of tef, oesophagocoloplasty and died with gastrooesofagostomy). the late mortality . % ( ) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il- , il- ) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, children ( months- years) were admitted in our picu in with the following diagnosis: bacteriemia ( ) sepsis ( ) and septic shock ( ) according to jacob's r f criteria. measurements: il- , il- , tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, , , hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il- , il- and tne high levels of tnf and il- are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd ( %). in patients pd lasted less then one week and there were no complications; in patients it lasted - days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. patients ( %) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles ( %, %, ~ %, %) of the most important pulmonary parameters were determined non-parametrically in weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of - healthy newborns ageing between - days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised % scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. ; : - . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during year period. as the validation study started on july , data collection is still on going and validation analyses will be carried out on july . up to now centers recruited cases. at present, characteristics of the sample recruited are the following: most of the patients were male ( %); the mean age is years with % of patiens having less than days; more than half were medical cases ( %) admitted from emergency room or from hospital floor ( %); % cases were admitted with an organ failure while % to be intensively monitored. icu-mortality was l %. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from consecutive paediatdc icu admissions over a year pedod. a proportional weight (pwt) was calculated as a percentage from the th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors ( . kg and . kg medians p = ) a lower pwt ( % and % medians p = . " . the incidence of malnutdtion , in our icu population was %. the mortality of manoudshed patients was' significantly increased (p = . ), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from . to . ). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample . , az validation sample . ). the modified formula is: legit = - . + . *prism score - . *age + . *weight category, where the probability of mortality is exp(iog/t)/ + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to ppm inhaled no in patients( m, f, median age . months, range day to years) with signs of increased pulmonary vascular resistance, there were pre and postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a % reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders( / ) were characterised by left to right shunts or pulmonary venous hypertension( / ). patient# was weaned from ecmo with inhaled no. patient# , without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient# had a pulmonary hypertensive crisis which responded to no. all non-responders( / ) had evidence of anatomic obstruction to pulmonary blood flow (# , , )or a low pvr(# ) on subsequent cardiac catheterisation. in patient # , lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these years, the incidence of sdra was . % of the total of admissions. the most common etiology was meningococcic septic shock. since , there is a decrease of its incidence. (from % to %) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was , years ( % males, % females), total mortality by sdra was % and there is an increase up to % since mean time of stay of the dead was , days and , days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: cases of severe traumatism in children admitted to our icu in the period from january to june were reviewed. age of patient ranged from months to years, % were males. in our series, % of cases suffered traumatism due to a traffic collision and % had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in % of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: % of our patients suffered cranioencephalic traumadsm (ct); in % it was an isolated picture and in % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in % of cases. % of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was . %. in . % important sequelae remained. all of these patients presented tepas on admission equal or lower than . % of those with traumatises had slight sequelae. . % of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation % of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only % in spite of a ventilation of % of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m to nun h and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to ca/of the total pulmonary perfusion. after days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a year old girl. results: a total of children with ards were treated with bovine surfactant (alveofact| cases were evalable. the median age was . years (range weeks to , years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of clays (range - days) after first symptoms of ards witha median doseof mg/ kg (range - mg/kg). in patients doses of surfactant were applied. during the hour before therapy, the median pao / fio -ratio was - . within min. after application of exogenous surfactant the pao / fio -ratio increased to with successive decrease over a period of hours to . accordingly, an increase in pao and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. of treated patients survived ( of the , respectively). of the surfactant doses were applied in the surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of - hours. the effect often could repeatedly reproduced, in one case after applications. the increase in oxygenation often allowed the reduction of fio and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with patients detected antenatally and another diagnosed at months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a year period patients under y of age fullfilled diagnostic criteria; were detected prenatally ( - weeks) and one was diagnosed at age mo. the fetuses had intermitent svt during gestation. all of them had pjrt in the first month of life at rates between and bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in to days. one was in sinus rhytm until age y; he then showed persistent pjrt over % of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over % of the day (follow-up ran and y). the mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction %) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to ; rfca was performed at m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient ( to see) recovery of sinus rhytm. ff,s clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients ( %) died. these were meningitis, head injury, sub-arachnoid bleeds, status epileptieus, leukaemie, drowning, and multiple trauma. calculated from the a admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from - %. of the deaths, i were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p % & %) survived. the median length of stay was days (range - days). patlents( %) received ventilatn~ support and patienta( %) were transferred to specialist units ( neurosciences, liver, cardiac, bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths ( of ) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, m and y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was and bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of ~ there was a gradual decrease in heart rate in the following hours ( - bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in to hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september to december , a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n= ) were treated with et. in total newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was . ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of newborns, treated for culture-positive sepsis in the same icu during the first eight months in . students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: death of cases during the study period, compared to deaths among the controls (p< . ). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o. ) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for days. a significant increase of granulocytes was ever noted already after the second administration until hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, months old, submitted to a neurosurgical interventiol after days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after days an intrathecal administration of vancomyein m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january ' till april ' , all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied ( females, males)i out of which doctors, residents and nurses. it was considered sign!ficant as mild bo, a score between - and as severe, the score of bo >. . results: subjects ( %) resulted positive for bo, out of which were females ( %) and were males ( %). the subjects with mild bo were / : was a doctor, residents and nurses. the subjects with severe bo were / , out of which resident and nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses ( subjects), as well as residents ( subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of ~tc-dtpa from the lung. methods: three groups of white sealand rabbits (bw . kg) were anesthetized, tracheotomized and paralyzed. groups were ventilated for six hours at pressure regulated volume control, set to deliver ml/kg with a frequency of /rain, i/e ratio = : and peep = cm hzo using a modified servo ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either / or / [no (ppm) / fioz]. after six hours of ventilation in these groups and immediately after anesthesia in group (control), ~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for minutes, monoexponential curves were fitted to the data and the clearance half-time (t was calculated. the t~/ mean • sd of the different groups were: t~a (mean -sd) h"e,i witl~ arf : di.ff:erent kinds, aged .q-ore mon't.hes to [ gears o : (bodi weight .~rom ., to kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se... ~ .ards'- ; :~,;,,arf o~ ::entral genes:i s .- , ,~ :inc lud ing men ingeenceph it :is- ~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.." ). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba- 'i~ier was. about . tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to dayns.,~ < .-:in , "t -irl lo;and> davs'-in 'l~atierr~{s i'i"ai s:ltiol~ o ; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to - ml~ar, fi ~ecreased to , . lind less with a = /,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th f'f'v, teoph :i. : . l:i.r~ (is- .mg/kg/day), g lucecdr t icostei~oids ( .... :~;mg/kg/day), when r exceeded in , -.];, times normal va i tea the e aqes/,'!:l"oln ~j,, ite :i.~;::.!;, ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom ! . '.' i', to !..'; , - , mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i. e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl of: . health children (asa i, ii) facing single uncomplicated surgical elective procedures . various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones . unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods - ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure > % systemic pressure, critically depressed right, ventricular function or an oxygenation index > . assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in situations ( patients, age days- , years), ino was applied - h postoperatively. oxygenation was improved in situations from _+ to + mmhg pc ; pulmonary pressure was reduced in situations from -* % to _+ % of systemic pressure. in situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume + -* %, cardiac output + -* %). in situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n= ], multi-organ-failure [n= ]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under months of life belonging to asa group . they all had been anaesthetised in . in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in cases we tbund: lafingospasm during induction in anaesthesia ( , %), bronchospasm during induction in anaesthesia ( , %), impaired intubation ( , ~ postanaesthetic laringospasm ( , %), supposed aspiration ( , %),postanaesthetic inspiratory stridor ( , %), postinductional inngoedema ( , %), death after months in consequative of infection pneumonie ( , %), none of these disorders was correlated the prematurity, infants suffered of post anaesthetic apnea, of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb ) were investigated dudng mechanical ventilation over a pedod of hours. minipigs with hemorrhagic shock were intubated and ventilated for days in an animal intensive care unit (icu). after hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of hours of types of filters showed an increase in weight of + % and airway pressure. bactedal celonisation ccured in nearly all fillers ( of ) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: patients in respiratory failure, age range weeks to years in a total of patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from hours to days. indications for use ef the device were: ) to facilitate weaning from ippv ) prevent reintubation of patients following unsuccessful extubation, and ) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao :fio ratio after cnp and eov-nb (p < . , and p= . respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco both with eov-nb and cnp (p= . and p= . respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p< . ) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao :fio , the fall in paco and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: women with ~h-gestosis (i group) and somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was % in the ist group and % in the iind group; gaseous metabolism - % and %, respectively; central hemodynamics was ~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, ( males and females; mean age . • were enrolled in the study. all patients presented criteria for pneumonia with pao /fio ratio ~ and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = em h ; peep = emh ; trigger = -lemh ) continuously admires" tered ( ' before fob fio = . ; during fob, fio = and for ' alter fob, fio = . ). pao /fio ratio as well as saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis ( ), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: mechanically ventilated patients, male and female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia ( ), ards ( ) and septic shock ( ); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: consecutive patients with severe ards (murray-score > , ; pao / fit < mmhg; male, female, mean age years) were conventionally ventilated (pcv, peep - mbar, i:e=i:i, ppeak < mbar). if after hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every hours. beside ultimate survival, parameters investigated were aado , pao /fio , and venous admixture (qs/qt). results: during the first hours in prone position of patients showed a significant decrease in qs/qt ( . % vs. . %) and aado ( vs. mmhg), and an increase in pao /fio ( vs. mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than hours before first application of prone position. after an average of position changes ( to ) of patients could be weaned from the ventilator. patient could leave tile hospital. i the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co , and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the % of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < mbar, ppeak < mbar) pulmonary function does not improve within - hours prone position should be applied. when after - position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed % for h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged ,g • days in patients with ardsvs , _+ l, days in control . mean staylcuwas lg _+ ,g days in the ards group vs , • , days in control group postoperative mortality rate was % in ards patients vs , % in those without respiratory failure. -ards incidence in liver transplantation is low ( , % in our sene) but it causes high mortality ( %) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (< kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio of . , peep of cm h , imv of bpm, it of %, and tv of ml/kg), sheep weighing . ~ . ) kg had lung injury induced by instilling ml/kg of . n hc into the trachea. ten minutes after injury, sheep with pao < ton" were randomized to continue gas ventilation (control, n= ) or to institute page (n= ). page was instituted by instilling . l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, minutes after injury, minutes after injury, and then every minutes for hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age _+ y;fev~ . _+ . l) and ards patients (age _+ ; lis . _+ . ) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao , svo , hb and methb were measured (abl ,osm ). mean intratracheal concentrations of no and no were continuously monitored using a chemiluminescence analyzer (nox ) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no ppm, no ppm and basal . after a steady state of rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a % increment in pao . results: papm was similar in both groups and decreased significantly after no (ards, basal . _+ . mmhg, no . + . mmhg, p < . ) (copd, basal . _+ . mmhg, no- . _+ . nrmhg, p< . ). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio _+ mmhg) vs ards group (paojfio _+ mmhg)(p< . ). after no- , pao increased ( _+ mmhg to _+ mmhg, p< . ) and qs/qt decreased ( + % to _+ %, p< . ) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao /fio was not correlated with basal paoflfio . no responders were / ( %) in ards group and / ( %) in copd group (p< . ). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis / . objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest ; : - ) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co elimination in different clinical conditions. methods: we have studied adult patients ( controls, acute respiratory failure, severe ards and copd) mechalfically ventilated (servo and c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco /vt] =facoz, vco vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of % variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at levels of vt ( . , . and . l) and four levels of peep ( , , and cmh objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied groups of subjects. n: normal subjects scheduled for minor surgery; arf: critically ill patients with mild acute respiratory failure; ards: patients with early ards (< h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne ( . l) a peep level of , , and cmh was applied in all patients. we calculated ers (cmh /l), rmin, c~rrs (cmh /l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co slope beyond vae (sipco in contrast to synthetic surfactant natural suffactants (alveofact| are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe , innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days ( , ). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated ventilated patients with severe ards (entry criterias: murray score > , ) in the ct scann of the university hospital. after a stabilisation period of minutes in supine position a thoracic ct scan slice cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) degree of left lateral position, b) degree of right lateral position. each lateral position was held for minutes. at the end of each of these periods a thoracic ct scan slice cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) ( ). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: %; l: %) as well as a significant decrease of the noninflated compartment (s: %, l: %) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: %, l: %) whereas the noninflated compartment increased significantly (s: %, l: %). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who required no inhalation with a dose of ppm for at least days. entry criteria for no injaalafioa were murray score >i . aud pat/fie < nun hg with peep >~ em i~o for at least hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox , see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least % and was stopped when the change in pao /fio ~ was below % or when the patient presented a paojf > mm hg a~er minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao /fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was . +_ . years and mean lung injury score was . • . . mortality was % ( / ), metahemoglobinemia . • . %, and no concentrations zero. paojf~o always improved significantly al~er ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > . , and a pao /f~o < with peep ~ cm h for at least h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were men and women with a mean age of . years ( - ) and mortality was % ( / ). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to emh with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr ; : - ) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a ml v r with a respiratory rate of /min and i/e ratio : ; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who were treated with no ( ppm). the onta'y criteria for no inhalation were murray score >/ . and paojfo z < mm fig and peep >/ cm i~o for at least hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between and ml&g, with constant inspiratory flow, respiratory rate was - /rain, and i/e ratio between : to : . all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox , see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo above % was observed after the inhalation of ppm no (group r) . when the cha~age in paojfi z was below % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ %) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. patients (age: - years; males) with no serious complications al~er mitral or multiple valves replacements and with more than hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head # ; differential pressure transducer pm- -tc "statham" w amplifier "kistler ") and flow-volume recording of forced expiration (fleish head # ) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on ( nay) and ( day) days. airways specific conductance (sg aw) has been calculated as a mean of - consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at and % of vc on - f-v curves (mef .~ , mef ) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of patients ( • years) presenting with acute exacerbation of copd (fevi: • ml, paco : • , ph: . • . ) and nonmvasively ventilated (pressure support through a full-face mask) between april and may twenty-seven ( %) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while ( %) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score ( • vs • , p = . ), creatinine serum concentration ( • vs • gm/l, p = . ), blood urea nitrogen (bun : • vs mm/l, p = . ), age ( • vs • , p = . ) were higher and encephalopathy ( vs %, p = . ) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = , p = . ) older than years (or = , p = . ) and presenting with bun >_ mmyl (or = , p = . ) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco , pao , fev ) were unable to predict tile failure of niv. conclusion : copd patients older than years, presenting with acute exacerbation, encephalopathy and bun > ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december we studied pa_ timnts, males and females(mean age +/- ); of the se had emphysema,lo chronic bronchitis, dilatative car diomyopatia,with tracheostomy and emphysema.mean pac at admission in icu was +/- mmhg,while when weaningbegan, +/- .mean autopeep was cmh ( - ).all patients were ventilated in crpv as long as four hours to calculate st tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep cmh objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond hours after trauma), is more insidious. the clinical and radiographic picture has to clear within to hours. ( ) methods: all headtrauma patients admitted from january to december , in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao -fio ratio < . results: neurotrauma patients were admitted during . patients ( %) presented with severe head injury (gcs< ), patients ( . %) with moderate (gcs - ) and patients ( . %) with minor head injury (gcs - ). overall mortulity was . % early (within h. after trauma) and delayed onset respiratory incidents were distinguished, counting for ( . %), respectively patients ( . %), patients ( . %) had early and late respiratory complications. early respiratory insufficiency was caused in patients ( . %) by aspiration, in patients ( . %) by lung contusion, in patient ( . %) by fat embolism and in patients ( %) by npe. in the late onset group patients ( . %) presented with pneumonia, ( . %) with fat embolism and ( . %) with npe. the npe group, patients, presented as follows: patients ( . %) developed early npe, and ( . %) delayed onset npe. patients ( %) died within the first days after admission, showing high mortality. gcs was less than in patients ( . %), indicating severity of head injuries. conclusions: high incidence of arf with various etiology ( , ~ was found in this population. in about % of all admitted hcadtrauma patients ( , % of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of em h with demand-flow continuous positive airway pressure (cpap) cm h . after a minimum of minutes on the above sehiogs: gastric intramucosai pc'o , abg, and a p . were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for hours for any reason. results: twenty patients met criteria and were studied over one month period in october . six of the twenty patients ( %) failed weaning. the mean and standard deviation is outlined in failure . +/- . . +/- . . +/- . . +/- . comparison between roc areas shows phi and p . to each show a statistically significant difference from an area of . (p<o. ). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #' . results. of the newborns referred to us for ecmo-therapy developed barotrauma, of the required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates , d- giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in ards patients mechanically ventilated for - h (mean murray lung injury score [ ] . + . ). patients were randomized to receive either first no and then pgi (n= ), or vice versa (n= ), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi were compared to pro-and past-challenge values. no (mean dose . + . ppm) increased the mean oxygenation index (pao /fio ) from + to + mmi-ig (p < . ) and reduced the shunt-flow from . + . to . : . % (p < . ). aernselized pgi (mean dose . + . " ng/kg rain) augmented pao /fio from + to + mmhg (p < . ), and decreased shunt from . + . to . + . % (p < . ). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in patients, both no and pgi caused an increase in pao /fio by at least rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from . : . to . : . mmhg in response to no, and from . : . to . : . mmhg (p < . ) in response to pgi . cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co -removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since , patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf , pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: ) in mean, platelets dropped from /nl to /nl within h after onset of ela. ) tat levels were already elevated before ela start ( ug/]) and demonstrated an additional peak h after onset of ela (up to ug/[). ) in parallel, there was a transient peak of pai-i levels (from to au/ml} h after onset of ela. ) in mean, fibrinogen levels dropped from to mg/ ml within h after onset of ela. ) in mean, cl-inhibitor levels dropped significantly from % to % after onset of ela and reached the initial levels within h. ) after membrane change, there were significant changes for c -inhibitor, fibrin-d-dimers and tt. ) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep ( - - - - cm h ) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was - _+ mi (x _+ se) or a - . _+ . % error. increasing peep from to cm h induced a + ml change in eelv. a difference of + ml ( . % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was _+ ml/ min (fig.i) . validation of calibration showed a mean error of -+ . % after rain. the new rip had a higher drift when cold (cold + ml/ min vs warm _+ ml/ min), whereas the old rip was thermally stable (cold - + ml/ rain vs warm i + ml/ min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo ) values is higher than suspected. objectives: determine the incidence, the significance and the pro nosis value of those differences,if they exist,in severe head injured-patients. material and methods: severe head-injured patients, coma glasgow scale (gcs) , with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than hours, were studied. data from bilateral sjo were obtained simultaneously, every hours. discrepancies were considered significant, when differences in sjo were > %. no chan es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: men and women were studied, aged • yrs. at arrival at hospital, gcs were < in and ) in to. the incidence of high icp() mmhg) were sz at the entry. the mean therapy index level required to control lop was ~l all patients required vasopressor therapy to maintain upp over ds mmhg. in patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo . in the others , sj were intermittently controhed.the mean time of monitoring were d. • days. ten patients died within this period. a total of . blood samples were analized. at arrival, sjo discrepancies were found in patients, b %. at hours, the incidence were lower, / , . %. at th day, were h/ , z and at day , when the catheters were retired, ii[ , z showed discrepancies. the ct showed new injuries in g z of patients with differences > ~ in sd values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo values and sjo differences. conclusions: the incidence of discrepancies between sjo was higher than expected in severe head-injured patients. these situation could reflect disturbances between demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied healthy, non-atop• nonsmoking subjects ( m/ f, + yr), which were pre-treated with s ( ,ug) or placebo, with a randomized, double-blind, crossover, design, before paf ( ,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every min over a min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from to x /l), and pmn(from to _+ x /l), and increased aapo (from . _+ . to . + . mmhg, p<o. ) and rrs(from . . to . . cmh .s.i q ) (p < . each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r = . , p < . ). by contrast, after s, paf did not induce any change in white ceil count (from to + x /i), pmn count (from to x /i), aapo (from . to . + . mmhg), and rrs (from . . to . . cmh .s.i ). compared with placebo, after s there was a lower ~lln-pmn lung activity ( . . vs . . cts/mci/pixel, p<o. ), lower intrapulmonary pmn retention ( . . vs . . %, p=o, ), and a faster washout ( . . vs . . s ~, p= , ). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)> . - . has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied failed weaning trials in copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco > mmhg from baseline accompanied by a fall in ph< . ; a respiratory frequency (f) > /min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo pneumoeytes. sections of generation , and from bronchial tree were free of pathological changes. conclusion: alter h inhalation of p~ji no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation . objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = ) trauma patients with acute lung injury (ali), group if (n = ) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using . % marcain. the following parameters were analysed: pao , sao , paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao and sao , decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t pts. with ari (pao : , @ , kpa. paco : , i- , kpa, ph: ~: , , hco : , :~ , mmol/ , sao : , ~- , %) who were hospitally treated due to pneumonia( pts.),emboly of the pulmonary artery( pts.) and severe attack of bronchial asthma ( pts). among tham there were ( , %) males and ( , %) females, average age , ~: , years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during hours (sodium-na,potassium-k, chlorine-c , calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l o, the object of our investigation was a group of pts with massive pneumonias, males ( . %), females ( . %),mean age yrs.thirteen ( %) of them were smokers, ( %) nonsmokers. only pt ( . %) had pre-existing chronic respiratory disease, and ( . %) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in pts( . %),physicaly ~onchial breathing in i~s ( . %),white cell count onder x /l in pts( . %). radiographicly, bilateral massive homogeneous shadows were found in pts ( . %), onilateral in pts( . %),pleural effusion in pts ( . %). abnormal renal function was found in pts ( . %). sputum culture was positive in pts ( %): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in , , cases respectively. all patients had remarcable hypoxernia (pao range from , to , kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf intrapulmonary shunt) were measured at controlled flow. in dogs (treatment), data were collected at baseline, during long injury (obtained rain after intravenous administration of oa . ml/kg), and again after bos ( mg/kg intravenously). in dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < . vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar tension is measured by the application of the alveolar air equation in which the arterial pco is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of . . the purpose of this study was to estimate the effective alveolar tension (pao eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in normal subjects and patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po (pao (i)), measured from the alveolar air equation in which paco was substituted by the effective alveolar pco (paco eff) and with the alveolar po measured from the simplified alveolar air equation (pa ). this study is complemantary to previous work for the estimation of paco eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response and co analyzer. the method is a computerised calculation of the effective alveolar po quite similar to that of paco eff, obtained from the simultaneously recorded at the mouth expiratory flow, and co concentration versus time curves. results: the results showed a mean difference (pao eff-pa (i)) of - . kpa in normal subjects and - , in patients. the mean of the difference (pao eff-paq ) and (pad (i]-pao z) was much greater than . in all subjects. the limits of agreement for the difference (paozeff-pa (i))were - . to . kpa in normal subjects and - . to . in patients, while those for the differences (pao eff-pad ) and (pao (i)-pad ) were very large ( > - . to > . ) in all subjects. conclusions: the effective alveolar po is very close to the ideal one in normal subjects, tn patients pao eff may excessively deviate from pa (i) due to the observed significant difference between the alveolar/tidal volume ratio for o and that for co . the alveolar po measured from the simplified alveolar air equation (pao ) differed substantially from pao eff and pad (i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood -carrying capacity and increased affinity of hemoglobin for are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa >lo mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i . erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value • mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value . + . mu/ml in group i, . • mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i . we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, ( . <g-i < . ); and group iii comprises patients with severe ards (all score > . ). broncoalveolar lavage (bal) was obtained after infusion of normal saline at ~ to intubated patients and cooled immediately. cells were removed after mild centrifugation ( x g, min, oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of % acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than l]s. we have investigated patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was . l/s _+ . l/s. the maximal deliverable flow of l/s was obtained in of patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no ) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl ( ~g apob/ml) was incubated with cu + ( , ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from to ~m. oxidation of ldl was monitored continuously with conjugated diene formation ( nm) and hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to hour renal isehaemia following by hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (< . nmol/g tissue in sop versus . nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a -year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in patients ( %). at the hospital admission, pac was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years through , patients were admitted to hospital in acute asthma episode. there were ( , %) pts with complications; mean age of yrs; females ( %). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was yrs (range from months to yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from to days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during a total of patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco greater than , kpa and/or an altered state of consciousness. mean age was , -d: , yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was ~ /min and puts rate .d: bts/imn. arterial blood gases revealed a pao of , ~ , kpa, paco of , • kpa and ph of , -+- , . area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every hours the first day and every hours the second and third day. at the end of hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total patients, were finally included in the so group and in the po group.patients in so group and po group had comparable ages (p<. ).peep in both groups was comparable (=. ) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at h.(p<. ) and h. (p<. ).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at h. (p<. ) with respect to initial values.fifteen cases of ards ( . %) in the so group and twenty five cases ( . %) in the po group were identified. the time of onset of ards was _+ hours in the so group and + b hours in the po group.the final outcome was very similar th both groups : mortality of % in the so group versus % in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi ) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score > ) recived inhaled pgi at - ng.kg.min " using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after rain of pgi inhalation. results: short-terro p~i inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao ) increased from to mmhg in patient and from to in patient , the ratio pao to the fraction of inspired oxygen increased from to (patient ) and from to (patient ). venous admixture decreased from % to % and from % to % in patient and respectively. mean pulmonary artery pressure decreased slightly from to mmhg in patient and from to mmhg in patient . no effects on systemic haemodynamics were observed in any patient. conclusions: pgi inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated ards-patients (age yr ( - ) mean, range) during - . the lowest pao /fio -ratio was ( - ), the worst murray score . ( . - . ), icu-stay ( - ) days and hospital mortality %. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments ( - ). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) lowering cholesterol using iovastatin treating hypertension using nifedipine heart transplantation intensive care of ards-patients conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients ( ). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of . % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= ) to analyse the effect of different levels of peep ( , and omh ) on evlw during hpe; ) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in groups of dogs by inflating a foley catheter in left auricular to a pressure of - r~uhg. peep levels of , i or m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over kg) were randomly divided in three gmups immediately alter infusing via a central vein . ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n= ) : vt: - ml/kg; zeep. group :(n= ) : vt: - ml/kg; peep: cm h . group :(n= ) : vt: - ml/kg; peep: emil . (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi> kg/m ) we partitioned the total respiratory system mechanics into its lung ( ) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap ; : ) . at three different levels of peep ( , , cmh ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in - , selecting adult patients with arf defined as: intubation and pao /fio < mmhg plus an fio > . or peep> cm i . results. patients met the arf criteria: of them ( . %) underwent prone positioning (p+). prone position use began in the early phase of arf ( . • days from the beginning, range - , median ). out of p+ pts were treated with controlled ventilation (cppv or pcv), while were on assisted ventilation (simv+ps) and on spontaneous breathing (cpap). only pts were awake when turned prone, while pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght . • h, range . - h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least . mmhg increase in pao /fio : / patients ( . %.) were responders when first pruned. the pao /fio changes induced by prone position are reported in the figure. pao /fio increased when patients were pruned (*p< . ) and remained higher than baseline values when returning supine(*p< . ). paco remained unchanged. prone positioning was used at least twice in / ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis . +. ) submitted to mechanical ventilation randomly received inhaled no doses in increasing or decreasing sequence: . , , , , , and ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_ cmh ) and fit (. +. ). the changes in vt and fit due to the no ( ppm in n ) injection in the ventilator external circuit were compensated for. results . the dose of . ppm, ineffective on papm, significantly improved oxygenation. the increase of pat and the decrease of q'va/q' and papm were nearly maximal at - ppm. no deterioration of arterial oxygenation was observed at no doses as high as ppm. co exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [ ] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no ( - ppb) in hospital central-supplied medical air. [ ] we have realised nitric oxide measurements in ten healthy volunteers, ( smokers and non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat ry limb of the ventilator. unfiltered central supply medical air contained : - ppb of no and - ppb of no , whereas central supplied oxygen was no/no free. samples were taken after equilibration periods of minutes, with increasing fit levels of . , . and . for subsequent minutes periods; paired values were recorded every s. the mean no value was . ppb (sd . ) and n o significant differences were found for different fit levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [ ] and that, previously reported, differences between smokers and non-smokers are not always striking [ ] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either rag (n= ) or rag (n= ) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed , , and rain after the start of nebulization. results: compared with mg regimen, mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of ng of salbutamol is not more effective than rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of neutropenic patients (wbc < /mm and/or pmn < /ram ), admitted to the icu between and , were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients ( . %) had a h~ematologic malignancy, and ( . %) a solid tumor. fifty-nine of the patients died ( . %); the mortality rate did not differ between both groups ( . and % respectively, p = . ). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < - ) and cardiovascular (p < - ) failures, and the number of organ system failures (p < - ) are associated to the risk of death. the saps (p < - ) and saps ii scores (p < - ) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = - ); neither the features of the underlying malignancy (p > . ), nor the duration of neutropenia before admission in icu (p = . ), nor the severity scores figs ii: p = . ) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co removal. generally, this has been used for short periods of time (< h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used ( % of the patient's cardiac output), sufficient to achieve both co removal and oxygenation. satisfactory gas-exchanged was accomplished (pco = - mmhg) with nearly total lung rest for a prolonged period ( h). however, the long ecmo duration was associated with two severe complica-ti ns: ) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and ) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, : ; ) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt ), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects ( male, -+ yr) and copd patients ( male, -+ yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial - min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was < %. lung function tests were: in normals (mean-sd), fevl%pred = • fevl/fvc%= -+ % , and in copd patients, fev~%pred= __. and fevi/fvc%= --. %. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in , ~ os patients; inconsiderable improvement -in , ~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e , h and , ~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease ust afte~ the course o~ t~eatment ( , h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s months in , ~ os patients, till one yea~ in ~ ~). in , ~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in , ~ os patients a period os ~emission lasted s ha]s a yea~. , ~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: mechanically ventilated patients ( copd, ards, other pulmonary diseases) were studied in two phases: ) during the acute phase of respiratory failure; ) during recovery - days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with - cmh ) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase ) or for two hours when they could sustain sb indefinitely (phase ). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. . during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. . inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit pg/ml)in balf (lavage of x ml in the right middle lobe). results: lif was not detected in the balf of healthy control patients and in only one ( pg/ml) out of patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of out of patients with full-blown ards ( + , mean + sem, range - pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r: . , p= . ), albumin ( r: . , p= . ) and protein level (r: . , p= . ). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, ; : - ) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which % of a tcg~-labelled test meal was eliminated from the stomach (t / ). baseline data (do) were recorded after enteral nutrition reached to ml daily. scintigraphic measurements were repeated days after cisapride ( ml orally, q.i.d) had been added to this regimen (d ). patients were considered cisapride responders when gastric clearance improved by more than % from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be + min. in healthy volunteers (crit care med, ; : - ) . five patients responded to cisapride (t / : + rain vs. + min at do and d , respectively) and five did not (t / : + min vs. _+ rain at do and d , respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> ml) gastric residues, vomiting (> times/day and abdominal distension) which disappeared in of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove , czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval patients were enrolled in the study enteral feeding was begun within hours of injury in trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ l short bowel syndrome) and group b (intensive care, surgical patients). all patients likely to require pig for a period of days (group a) or days (group b) were studied. the pn regimens administered were the following : combination with g of mct/lct fat emulsion end , g of nitrogen, in liter end glucose requirements were met by imfizsion of l liter of glucose - % via a "y " connection. lipid thus provided % of the non introgen calories. total daily calorie intake was to ] kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf- , gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md , chiolero r md , pannatier a phd , berger l , cayeux c , voirol p , hurni m md . surgical icu, pharmacy, and cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, patients, aged _+ years (mean_+sd) were assigned to groups (anaesthesia: fentanyl gg/kg + midazolam): group (n= ): reference group, with normal hemodynamic status, easy recovery. group ('n= ): patients in low output syndrome, cardiac index < . i/m on day (d ) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol g, was given intragastrically on d + d : plasma levels measured (h.p.l.c), at administration (to), t - - - - - and rain. hemodynamic status assessed with pulmonary artery catheter. healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d in all patients (no difference between groups). on d , peak paracetamol level was significantly lower in group (low cardiac output): in group the area under the curve on d and d were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d in all patients, and reverted to normal between d and d in case of normal cardiac function, but not in case of low output syndrome. the decrease on d can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [ till november, there was carried out the randomized study of patients with general purulent peritonitis; among them persons constituted the control group and -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega- fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of . (std. dev. -+ . ). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg , hospal,uk, lactate concentration retool/l). date obtained hr before and - hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow ml/mlu, dialysate solution flow i /hr), and approximate daily lactate flux into the patient was calculated to be as high as mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels ( . + . axtd . + . ,respectively; p< . ,paired t-test). when patiente with increased basal lactete (~- ) were compared to paliente with normal basal values (n= ), no difference in laotete increase was fmmd (p= . , manova). patiente with severe liver dysfunction ( points in mop scomlg, n= ) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste ( or point in mof scoring, n=ll), rite values being . + . and . + . , respectively (p< . , student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p= . , manova). in pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t =o.ol; p--o. ) or oxygen consumption (reversed fie, k) (r -q).o ;p-- . ) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of polytraumafized patients, split in two groups of ten patients each, with mean ages of _+ an -+ years. due to their condition, all patients required p.n. for at least days. both groups were subjected to isocalorie and isonitrogenous solutions ( ci/kg/ day and . g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a % concentration and solution b %. blood samples determinations during days , , , after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < . ) and prealbumin (p < . ). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < . ). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p< . ) and prealbumin (p < . ) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < . ) when considering the interaction of both the type of diet and the time course of pn. conclusions: . solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. . no significant differences on plasma proteins were found between the two solutions having % or % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids g/i; glucose/xylito g/ g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids g/i; glucose g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on patients with severe brain injury. there was no difference in body mass index bmi (group a: . +/- . kg/m and group b: . +/- . kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started - hours after trauma or surgery with carbohydrates and aminoacids. fat was added h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for days in both groups. serum tipase steadily rose to lull in group a and . lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a -fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design groups (n= each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct- %), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct / - %) and group iii placebo (control), during h ( mg/kg/min each). we measured before, at the end of h infusion, and h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, -keto pgf~,, and ltb ). at the end of the fat emulsion, groups (i and il) to , • to , • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly ( . % and %) (p< . ). eicosanoids were increased at baseline compared to reference values (p< , ). a decrease (p<o, l in the arterio-venous difference of the vasodilatador prostanoid ( -keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of / in group i (n= ) , and a carbohydrate/fat ratio of / in group i] (n = ) during consecutive days. indirect calorimetry was set at baseline and hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day of enteral feeding. urine was collected during hours and was used to measure h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose -phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after h of nutrition patients in group i showed an increase in carbohydrate oxidation (from . % to . %, p= . ), and in group ii a decrease in protein oxidation (from % to %, p= . ). after days of enteral feeding a tendency to increase in glycogen concentration (group i, p= . ; and group ii,p= . ) was observed. while patients in group i showed a tendency (p= . ) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p = . ). in group i, the pao /fio ratio increased after days of treatment (from . to . ; p= . ), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in healthy humans (control) and in patients admitted to the intensive care unit (icu) at days , and . the apache ii of the patients studied was _+ . there was an increase of uer in the critically ill patients compared with controls (o. _+ . vs . _+ . ) (p< . ). in contrast, no changes in uer between days , and were observed ( . -+ . . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r= . ) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl /av). patients were divided for days of en according to the presence (group ) or absence (group ) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p< . for statistical significance. (vr%) group (vr%) tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: ) abdominal distension (abd), ) increase of gastdc residuals (igr), ) vomits (vom), ) diet regurgitation (reg), ) diarrhea (dia) and ) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t gr glucose/ h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only gr/ h glucose was provided, the mean values of vco output were _+_ and !-_ ml/min respectively. there was no difference in vco output between with low carbohydrates and high fat regimen and standard regimen, mean values were t . -+ and _+ respectively.the high calories regimen and tpn resulted in higher vco output, mean values _+ and • respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco output, in the contrary, high calories intake as well as tpn increased vco output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b raud j.j. m tabolic intensive care unit and nephrology department. lapeyronie hospital. montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january to january . patients (pts) (male: , female:lo; mean age: + y.o.) were admitted with rh defined as cpk> iu/ . etiologies were: traumatic and ischaemic , infectious , toxic , excess activity . factors studied were: simplified acute physiologic score (saps: . + . ), organ systemic failure (osf: . _-!- . ), diagnosis delay (d: +_ h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a hours period. arf was observed in pts. in non-arf and arf groups respectively, saps ( . _+ . vs . + . ), deshydratation ( vs ), sepsis ( vs ), phosphorus ( . + . vs . -+ . ), calcium ( . + . vs . _+ . ), ward score ( _+ . vs . + . ) were significantly different. however, no significance was observed in uar ( -+ vs -+ ) and ci ( _+ vs _+ ). patients required hemodialysis (hd) ( : sessions) and remained dialysis free. only osf ( . _+ . vs . -+ . ), ward score ( . _-/- . vs . _+ . ) and ci ( +_ vs -+ ) appeared significantly higher in pts requiring hd. pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over ) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x . . tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a hr infusion were investigated. methods: after approval by the local ethic committee healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using , -d glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor ( . pg/kg/min) and dob ( pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ ~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p< . , anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a / -adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet ( patients) or an isonitrogenous isocaloric parenteral support (tpn) ( patients) (total energy: kcal, nitrogen: . g, carbohydrates: g, fat: g, n/non protein calories: / ) at least for days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day in the tpn group and by day in the enteral group ( . % of the enterally fed patients and % of the tpn patients maintained in positive balance the day of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p= . ). thyroxine-binding prealbnmin rose significantly in both groups as well (p= . and . respectively). statistically significant rises in lymphocyte counts (p= . and . respectively), in levels of c (p= . and . ) respectively), iga (p= . ), igg (p= . and . respectively) and igm (p= . ) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group ( . %) and the tpn group ( %). by the end of the study the incidence of negative responsiveness was . % and . % respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in . % of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (> ml/ hours) fasting gastric aspirates. methods : antral ( sites separated each other from . cm), duodenal ( site) and jejunal ( site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder ( a recorder, hewlett-packard) then anal) ,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), ) presence of bursts (> min) of nonpropagated phasic pressure and ) presence of sustained (> min) uncnardinate pressure activity. patients with a volume of gastric aspirates of • (sd) [median ml/ hrs were investigated for - [median minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n= ) or migrated abnormally (retrograde propagation : n= ; retrograde and stationnary : n= ) in pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in pts and sustained uncoordinate pressure activity was found in pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n= }. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included patients with hypokalemia in whom rapid potassium replacement ( meq kci in h) was performed: patients received mg sulfate ( g in hours) and patients received a corresponding saline infusion. measurements were made at time , + , + and + hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time and h (p < . -students-newman-keuls). (table ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the " authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients . aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a months period and requiring artificial ventilation and enteral nutrition for a period of at least days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin ( rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. patients (male/female : / : mean age : . + . years; mean apacbell score : .t • . ) wore enrolled into the study.the procedure was performed within the dab,s following institution of mechanical ventilation. jejunal access was obtained in all patients without resort to enduscopy in , • . min.(total duration of the procedure). mean duration of fluoroscopy was . + . rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow % of normal values in or more of the above criteria were considered undernourished (und).statistical analysis was performed using % analysis.statistical significance was established at p<o.o . results: a total of patients( female, male) -with a mortality rate (~) of , % -were studied, aged -+ years and a median ]enght of stay of days. -nourished (nou) at ad and at dis - ; ~ %; -nou at ad and und at dis - ; ) , % ; -und at ad and und at dis - ; ~ . %; -und at ad and nou at dis - ; ~ , %. we observed a p< . when we compared groups and (p= . ), and groups and (p= . ). variation in nutritional status and longht of stay: -nou at ad and nou at dis = > median lenght of stay days; und at ad and und at dis = > median lengbt of stay days; nutritional status and age at admission: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) nutritional status and age at discharge: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) we observed a p<o. when we compared groups and (p=o. ) and groups and (p = . ) conclusion: such study permits to conclude that most of our patients ( . %), staying in icu for more than days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf- levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient baseline hlood samples for circulating gf-i were obtained over a hour period, prior to il!iection of p.g/kg of rhlgf- subcutaneously. a further l samples were taken fi'om each subject over the next hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age ( - ) years, apache ii score ( - ), and length of icu stay ( - ) days. baseiine circulating igf- levels were low ( < - ) ng/ml reaching a peak of only ( < - ) ng/ml. of the patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining patients tmax was at ( - ) hours, clearance was ( - ) ml/min, vd was . ( . - . ) l/kg, and tia . ( . - . ) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients ( . vs . l/kg), but clearance was greater ( vs ml/min), tmax earlier ( vs hours) and t~a shorter ( . vs i . homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january to march , patients underwent surgery for acute bacterial endocarditis: had native valve endocarditis and had prosthetic valve endocarditis. there were men and women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was + cc and in the aprotinin was :t: (p < , ). the mortality in icu was , %, the staphyloccocical endoearditis mortality was , % and the no-staphyloccocical endocarditis mortality was , % (p< , ). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality ( %). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: patients (ventilation period > days) were randomized and allocated to the sdd group (n= ) or the control group (n= ). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of rag polymi~ mg tobramycin and mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: smears were examined in the sdd group. mrsa strains were detected in samples ( . %) from patients, and in patients they were detected for a period of up to weeks. the positive smears were districted as follows: tracheal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal ( . %). severe mrsa-induced infections were observed in patients (infection rate . % of the colonized sdd patients). smears were examined in the control group. ivlrsa swains were r in samples ( . %) from patients, but only repeatedly over a period of up to days in patients. the po~tive snmars were distributed as follows: traclmal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal / ( . %). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip . g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: hospitalized patients have been documented in this multicenter trial during a year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > ~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip . g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n= ), followed by intraabdominal (n= ) and skin and soft tissue infections (n= ). % of the lrtis wvre nosocomial acquired and in % the treatment was conducted as monotherapy. in % the lrti was treated with taz/pip b.i.d, and in % t.i.d. pseudomonas spp. (n= ) and staph..aureus (n= ) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip . g b.i.d, and t.i.d, were % and % respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to . g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of cases of severe generalized tetanus (sgt), treated in our icu the last years. we review cases of sgt ( m, f), mean age . years. in eases the entry site of c.tetanus was a skin laceration, in case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first cases incubation period was short ( - days) and so was the period of onset ( - days). all patients needed mechanical ventilation (range - days), initally through an orotracheal tube,and later through a tracheostomy, performed • days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to weeks in the most severe cases. significant autonomic nervous system dysfunction was present in cases occurring - days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in cases. nosocomial infections occurred in eases, with sepsis and mof in one. average stay in the icu was - days. one patient died with severe septic complications and one was discharged with severe disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, out of examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi , intensive care unit, kb firule split spin~ideva ! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a -hour period.criteria for the suspected bacteremia were body temperature above c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in ( %) patients hemocultures was negative at both sites,whereas in the remaining ( %) they were positive.for twentyone ( ~ of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in ( . %) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than days in patients and for more than days in patients.from patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other had the catheter from - o days. we neither found significant differences in hemodynamic dates : objectives: , to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). . to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included consecutive ventilated patients ( male and female, _ + yr; apache ii score -+ ) over a period of months. et aspirated samples with and without saline were taken daily from day of intubation until pathogen~ were presented in counts of _> per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between to days. bacterial transfer between staff and patients was noted in % of patients until day of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was % with an overall mortality rate of %. acinetobacter anitratas (no ), staphylococcus aureus (no. ), klebsiella pna~moniae (no. ) and pscudomonas aeruginosa (no. ) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. . there is a close relationship between the microflora of personnel, patients and equipment. . bacteria transfer was noted both to and from patients. . strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to % of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from ~ to %. enteric gram-negative bacilli have been implicated in % to % of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for % to % of these pneumonias. importantly, epidemics of/ - actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than % of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and ;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from to ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i patients) or equal volumes of human albumin % (control group; patients). wig (sandoglobulin) was administered in a total dose of g/kg divided in a four time regimen on days , , and post-admission. three blood collections were performe& before the first dose (day ) and hours after the third and the fourth dose (days and respectively). complement, lgg fractions, the sba at ~ and at o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day ( +_ vs • ns and + vs + , ns), whereas they were significantly higher (p< ) in the v g group on days ( _+_ vs + , p< ) and ( _+ vs +i , p< . ). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas ( ~ c) at rain in ivig group vs control group were: - _+ vs - • ns for day , _+ vs - _+ p< for day and _+ vs - + p< for day . the mean (+sd) sbas ( ~ c) at rain presented a significant improvement over those of ~ c but for the control group remained negative a~d were respectively as following: -~ • vs - + , ns for day , +_ vs - _+ , p< . for day and _+ vs - _+ , p< . for day . the increase of temperature induced a -fold improvement of sba in iv g group and -fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p< ) in the ivig group than in the control ( vs , and vs , respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune , d- bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from the'overall resistance in intensive care units towards oftoxacin was %, whereas in other hospital wards the percentage of resistance was . %, in out-patients, however, only .$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid• in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of postoperative adults pts of mean age . + . years old, with a mean icu stay of . _+ . days, following an open heart surgery from july to april , pts ( %) remained in icu for more than days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole ( mg/day) ( patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of postoperative pts with prolonged jcu stay, pts ( . %) developed systemic candid• usually after the th postoperative day. they were males and females of mean age +_ . years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group ( min vs , and vs min). all these pts received inotropes per• (mean value= . ). during their icu stay, pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of days. the median icu and hospital stay was and days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: . a significant percentage of patients who remained in the postoperative icu for more than days developed systemic candidiasis. . all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. . fluconazole seems to be a very good alternative to amphotericin-b. . fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may to february . results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a -hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the th to th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis ( %), dry mouth ( %), dysfagia ( %), asthenia ( %), palpebral ptosis ( %), accomodation paralisis ( %) and urinary retention ( %). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an -month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high ( - %). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and month outcome. patients required mv; were immunocompromised (ic) including hiv. tb was pleuropulmonary in , disseminated in and meningeal in . results: mr was % in icu, % in hospital and % at month. / ( %) < . mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po /fio ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high ( % at month). need for mv increased mortality ( % vs %). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january tit march . in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total f pts were studied, males; mean age , _+ ( - ); apache ii score , + , ; glasgow coma scale (gcs) at admission , _+ , ; ( %) pts suffer from cronic pathology; ( %) pts diabetes mellitus (dm), ( , %) pts had had a previous cranial traumatism. in cases the source of infection was otic and also in ( %) episodes of pm there were bacteriemia. in out of ( %) pts that ct was performed no radiologic abnormalities were shown, of them presented cerebral oedema and pts a cerebral abscess. twenty-eight percent presented seixures, % hemiparesia, , % respiratory failure, , % shock, i % renal failure, , % multiple organ failure (mof). as for treatment refers , % pts recieved only penicillin, , % pts only tcg, , % pts tcg followed by penicillin and , % pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and , % vasoaetive drugs. the mean icu stay was , : days ( - ). twelve ( , %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the and hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at and hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of (} immunologycal parameters has been offered:t-calls total/cd +/;t-helper/cd +/;t-supressor/cd +/ th/ts ratio;b-cells/cd +/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd +mean= . %/ . %- . %/and cd +mean= . %/ % - . %/;inverted th/ts ratio ,mean=o. / . - , /;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population we prospectively studied pts, . % ( / -group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t and t type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in . %, tv in . %, av in % and pv in . ~ and was multiple in . %. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations ( % tr, pe %, mortality %). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type ,enterotoxin a and toxic shock syndrome toxin- .eight of patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from .futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from . these countermeasures for prevention of nosocomial infections after significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -- cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao : mmhg with fio : . , pco : mmhg, ph: . , hco : mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung / ). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was . mm , platelet: . ram , hematesrlt: %, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high ( / , negative<ill ) and iglm antibodies also very high (elisa, a= . , negative< . ) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period ( - ): group a, patients hiv+, and group b, patients hiv-. apache ii was identical in the groups (p=ns). group a required more often mechanical ventilation (p= ,o ), had a higher p(a-a)o (p= , ) and metabolic acidosis was more frequent (p= , ). regarding laboratorial parameters group a had a lower no. of linfocytes (p= , ), a higher ldh (p= , ) and a more marked hypoalbuminemia (p=o, ). mortality was higer in group a ( , %) than in group b ( , %), (p= , ). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p= , ). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o , metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, gr tid) plus netilmicin (netromycin, mg bid) (group b). a total of patients (pts) ( males and females, of mean age . + . years old) were included in the study over a period of one year; in group a and in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was . %; . % in group a and . % in group b (p=ns). pts ( . %) developed infection following cabg, pts ( . %) following valve replacement and pts ( . %) after other cardiac surgery. they were males ( . %) and females ( . %). endocarditis has occurred . % in group a and . % in group b. severe wound infection was recorded in . % in group a and in . % in group b. one case of sepsis ( . %) in group a and in group b ( . %). respiratory infection occurred in pts of group a ( . %) and in pts of group b ( . %). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in ( . %) in group a and ( . %) pts in group b. pts ( . %) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the patients with lpp admitted in our icu during the last years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . they represented . % of cap. seven patients were males and female, with mean age . + . years. tiss was . + . and apache ii . + . . all, but patient, were under mechanical yen tilation (mv) during a mean period of . • (min-l, max- ) days. two pneumonias occurred beyond the season, while patients had an epidemiological history. only patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in , renal failure in (due to rhabdomy~ lysis in ). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in patients. mortality rate was %. deceased patients had initially higher apache ii, (a-a) , and lower natriemia. comparing lpp with the other cap (n= ), both submitted to mv, mortality rate was similar ( , % versus . %). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > cfu cfu/ml. broncho-alv~lat lavage (bal) ~= c'fu/rnl or positive blood culture. were excluded for rapture of treatment ; were analysed (shift with oral antibiotic : ; prohibited antibiotics associations : ; resistant germ : ). clinical data : age , • , ; saps • , ; mac cabe i : , % -ii : , % -iii : , . , % of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in ( , %), copd ( , %), aspiration pneumonia ( , %). germs were isolated (psb , bal , blood culture ) : s. pneumoniac ( , %), h. influeazae ( , %), sttep~:occns ( , %), saar ns ( , %), enterobaetdrindr ( , %), mosexella catarrhalis ( , %), othem . / ( , %) were sensitive to freatment. the ltentment was mg/kg/d of ampiclllin and mg/kg/d of sulbactam in continuous iv adminisu'ation during at least days. clinical eff~ienev : success ( %), failures ( %) with superinfeetion , worsening or relapse , dead , side effects . there was no difference between etiologies : primiti~;e~ , %, copd , %, aspiration pneamoniae , %. the bacteriological effieieacy was evaluated only for patients with eradication ( , %), eradication but super~ection ( , %) : with pseadomoaas a&ogiuosa , eater~ac~ ; beeteriological failure ( , %). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > h were randomized to received topical application oga cl suspension ( %) qshrs, a cl gel ( %) q hrs or a placebo. in addition all vpts received a nasal and a op spray ( %) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n= n= n= n= / / / * / / / / * / / / / / / / / / i *p = , byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r enmmtion, * service des admissiens-urgeuces centre hospitalier g- ndral - saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a beds unit. the prospective study includes all the patients comn~ in from / / to / / . the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of nosoeomjal inthrtions: i abcess and bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed different periods: period ( / / to / / ) and period ( / / to / / ) for all .e peripheral catheters inserted in the i.c.u. period , % , % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo ) to measurements of the mechanical work of breathing (web) and p . . methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation ( - days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto , , , mbar far rain periods each. all patients had a peep between - mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display ( ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p . was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo and vco were measured using the pb metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p< . , compared to ips mbar. results: the values for breathing pattern, web, p . , vo and vco are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: episodes in consecutive patients (mean age: . years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, patients had nosocomial pneumonia, had urinary tract infection [ with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations ( - s cfu/ml) in occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over . mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p< . results: of the total admissions were fully evaluable. of them ( %) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for > h were compared: group = hme replaced every day, n= episodes of mv in patients; group = hme changed every h, n= episodes in patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~ cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every h. results: the groups were similar in terms of age, indication for and overall duration of mv ( +_ . vs +_ days, p= . ), and severity of illness (saps: --- . vs . +_ . , p= . ). the maximal values for peak airway pressure were identical in both groups ( . -+ . vs . • cmh , p= . ). obstruction of the tracheal tube was observed in only one instance in a group patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same ( / vs / , p= . ), as was duration of mv before the occurence of pneumonia ( • vs . +_ . , p= . ) and overall mortality rate ( vs , p= . ). conclusions: the clinical efficiency of this hme does not seem altered after days of use. indeed, replacing this hme every h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed patients (pts) admitted to our icu from july to december with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv ( cases), herpes zoster varicellae ( ), measle ( ), rabies ( ), unidentified ( ). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts ( / ) had an abnormal mri. among them, pts made a complete recovery, in comparison with / pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in cases (with poor outcome in one) and was improved in one. spect was found abnormal in / pts (among them, pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in % of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied pts, # ( , %), ( , %). hean age: , • years ( - ). mean stay in icu: , • days ( - ). they were divided in groups: a( pts) who did not suffer from pneumonia and b ( pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) times: on days o, and of the pneumonia for group b and respectively for g~oup a. conclusions: ) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts< years (p<o, ). ) the frequency of isolation of kp in icu pts during - was - %, but now ( - ) kp becomes more and more rare ( - %) and is isolated especially in elderly pts. ) its sensibility to antibiotics remains always the same. ) the prognosis of np in our study was independent from age (p<o, ), sex, presence of bacteremia (p< , ) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at and hours after the iv (bolus) or the et (nebulized) administration of mg of gm in seven criticcally ill patients ( with established renal failure). safety was defined as peak and trough plasma gm levels _< than and ijg/ml respectively and adequancy as gm levels in bronchial secretions _> , ijg/ml. results: gentamicin was administered by the et and iv routes in and separate sessions respectively. a total of samples were assayed, in bronchial secretions (bs) and in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route ( , + , vs , _+ , pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in % of patients after et administration, compared to % after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route ( , + , vs , • , pg/ml respectively, p _< . ). the et administration resulted in toxic bronchia~ gm levels in % of the specimens. % of these samples were from patients with renal failure, however toxic blood levels were reached in only % of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from groups of patients were tested. group i: patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of patients with a np without anaerobic bacteria, group ill: a control group of patients with dental stumps but without pulmonary infection, group iv: a control group of healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i ( • was statistically higher (p=o.o ) than in the control groups (respectively: + , _+ , _+ ). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t > . ~ c) in all adult patients admitted for >- hours in the icu during a two month period. these patients were studied for consecutive days. and werc classified in groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of patients studied, dec'eloped fever ( %). including (after complete evaluation) d, p and u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u ( . • versus . • and . -~ . ~ versus . - . , respectively p= . and p= . ). most common sources of infection in d were the lungs in patients ( %) and urina .ry tract in ( %). of these patients had positive blood cultures ( %). the overall mortality was % ( % in d, % in p and % in u. differences ns). antibiotics were given in % of d, % of p and % of u ( patients). in p there was a non significant lower mortality." in patients who received antibiotics ( / ( %) versus / ( %) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: patieet with a stay of more than days in inteaasive care were screened for candida infection. patients were treated with antifungal therapy due to either an increased antigen titre of -> : or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of : or higher. results: the median length of stay was (ranging from to ) days, the mean apache ii score on admission was (+_ . sd) points. of patients patients died ( . %). in the group treated with antifungnls ( patients) patients died ( . %). although of the patients only ( . %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher ( . % vs. . %, p < . [chi-square-test]). in patients an antigen concentration-> : was measured. seroconversion was found in patients. the most common fungus was candida albicans ( . %). furtberm re, candida glabrata was found in . %. most of the patients were treated with x mg fluconazole ( patients). in patients therapy was changed to amphotericin b/flucytosine. in patients therapy was started with amphotericine b and flucytosine. in patients a threefold decrease of candida antigen titre was found. patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied patients ( males and females, age + ) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at rpm for min revealed: i) more than leukocytes per optic field, ii) squamous epithelial cell less than percent and iii) one or more microorganisms per optic field on magnification. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap and patients did not. in cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was , -+ , , while in patients without vap was , + , . there was a significantly higher incidence of vap in patients who had i) coma (gcs < ) and ii) been receiving neuromuscular blockade (p< . ) . the sensitivity of the gram stain for vap diagnosis was %, the specificity , %, the positive predictive value %, and the negative predictive value , %. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october and february all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the psb performed for suspected mvp ( nosocomial and community-acquired pneumonia), yielded at least one micro-organism (positive psb : %). of positive psb demonstrated only aerobic bacteria and ( %) yielded with anaerobes. in out patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia ( / positive psb). strains of anaerobes were isolated. prevotella species represent out these strains ( %) the most frequent anaerobic species were prevotella oralis ( ) p. intermedia ( ) and p. buccae ( ). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied multi-trauma patients (mean age + years, apache ii . + ), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than and cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap, and patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)< (x = . , p< . ), the administration of neuromuscular blockade (x = . , p< . ), the duration of mechanical ventilation to be greater than days (x = . , p< . ), the flail chest (x = . , p< . ), the parenteral nutrition (x = . , p< . ), the ards (x = . , p< . ), the abbreviated injury scale (ais) of more than for thorax (:,: = . , p< . ), the pneumothorax (x = . , p< . ) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: . , p< . ), flail chest (f: . , p= . ), and gcs (< ) (f: . , p= . ). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (< ), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= , + , x~ + , x + , x , were x i-apache-ii score points, x -evidence of septic shock, x -endocrinopathy. the explanatory power of this quation was evidenced by roc of . , se (v - . introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in patients during the first week after trauma or the onset of sepsis. patients: group : (n = ) multiple trauma iss > , group : (n = ): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z- , pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. + . &-_ ( - ) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of granulocytopenie(wbc< , xl ) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in . % of patients before ss and in all patients after ss. only patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto ) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was . mt-m " , and le,~ and ie'i~ w~ . units in sic. we managed to maintain op of . - . ml.m " and ieto of . - . units in hst. patients from with mosf survived in sic and patients from survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and day survival were also recorded. biochemical data were compared with results from healthy subjects. results: xanthine oxidase activity was . + . units/i in patients (mean :t: sem) and . + . units/i in controls (p<o. , mann whitney u test), and was highest in the patients who survived (p< . ). malondialdehyde concentrations were elevated ( . • . prnol/i compared with . • . pmol/i in controls, p< . ). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco ) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p ) methods the pco electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography % of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on - % and general peripheral resistance increased on - %). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was - mg/kg during - days. as a result of such therapy was also the oliguric stage shortening (ac n ).in , % of the cases unsatisfactory datas of central hemodynamic,combined with high (more than cm h ) venous pressure,lung oedema.then nanipruss ( , - , mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton ( , - , mg/kg) and dopamine ( - mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during - days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; .ruptured aneurysm of the abdominal aorta (raaa) n = .elective vascular surgery (evs) n = .abdominal sepsis n - .trauma n = .others ( e.g. malignancy, obstetric emergencies) n- seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients % , group % , group % , group % , group % , group % . mortality increased with the number of failing organ systems; mortality for all patients with > failing organsysterns was % the only exception being the subgroup of trauma patients where mortality under these circumstances was o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( % ~ % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between and , and the function of , or organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol patients ( - years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: ) to increase the effect on microbic agents and the efficacy of treatment in combined infections; ) to lessen the possibility of the exciters'resistence to antibiotics; ) to prevent the development of superinfection: ) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was ( - ). when indicated, patients had blood volume measurements simultaneously based on the techniques of, i) dilution of ~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was ml ( - ml) compared with ml( - ml) with slcr labelled red cells (r= . ). regression analysis produced an intercept at ml. the slope of the regression line was . ( . - . , % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock ( - mmhg for rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of . mg/kg or . mg/kg at the end of resuscitation. results: infusion of . mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the h survival rate was significantly improved from . % in the control group to . % in the treatment group (p< . ). in contrast, the high dose of . mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest- ) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: patients have been hospitalized in our icu from january to may . , ( %) with mosf. among mosf pati~ts, ( %) have had hepatic dysfunction defined according to hebert (bilirubin ~ ttmop , chest ). thirty six of these patients acquired hepatic dysfunction after admission in the icu. these patients were compared with mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< . are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao /fio , arterial lactate, do were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to amag/~ factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f tm [fresenius] , ct tm [baxter] , and filtra tm [hospal]) were assessed. each filter was changed intentionally after a hours" use. together the data of filters were evaluated, each at three different times (immediately after its onset [ hi, after h, and after h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p< , ) showing the f tm membrane excelling filtra mand ct tm the more. the loss of inuline clearance ( mi/min/m ) after h, however, was insignificant for all filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after h (p< , ), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was years (y), patients ( % ) were < y, patients ( %) were >= y. the incidence of dialysed arf in our mixed intensive care departement is %/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively %, % and %. treatment: cavh (cd) or cvvh (cd) was used in patients ( %), hemedialysis (hd) was used in patients ( %). data: mean apache scores were the same for cd and hd ( for both groups), patients treated with continuous dialysis techniques had significantly (p<o, ) more need for ventilation ( % vs %), elevated bilirubin ( % vs %) and a higher vasopresser need ( % vs %@ than patients treated with hemedialysis. there was a trend towards more sepsis ( % vs %; p= . ) mad coaguiation disorders ( % vs %; p= . ) in cd. taere were sign/ficantly more younger patients (< y) treated with cd ( % vs %, p< .os). mean apache score was significantly lower in patiants< y than as patienta>= y ( vs ; p< . ). patients< y had significantly (i}< . ) more coagulation disorders ( % vs %) and elevated bilirabin ( % vs %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd ( % vs ~ p< . ). patiants>= y had a comparable sr vs patients< y ( ") */e vs %; p----a.s.). tha global survival rate (sr) was % ( patients). conclusions : diaiysed arf has a well known lowsurvival rate ( %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache score couldn't illustrate that. patient~ y with arf have the same survival rate as patients< y: although patients >=- y have a higher apache score they have less organ faille. the avacbe score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < . initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of . %. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo . sao , diuresis, pha, and hemoglobin were repeatedly registered during the first hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: measurements of co were obtained, ranging . objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo ) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do /vo > its co> relation to other parameters of do /vo disturbance, and with postoperative complications and clinical course. methods: patients ( male, female) undergoing cardiac surgical procedures were included ( myocardiai revascularizations, valvular substitutions, constrictive pericarditis). mean age was + years, mean weight _+ kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period ( ', ', ", ' and h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo ), diuresis and arterial ph (pha) were also recorded. results: phi values ranged . to . (mean . ( . ); the mean values of clinical evolution were: extubation time, _+ hr.; discharge from postoperative care unit, - hr.; and hospital total postoperative time, _+ . days. complications registered were: perioperative acute myocardial infarctions, cases of respiratory insufficiency, occlusion of coronary bypass, an ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > . ) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo , pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: ( ) gutidrrez g: lancet ; : - . ( ) landow i: acta anaesthesiol scand ; : - . the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p< . . according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb< and b: >sg/dl: i: ac< . and ii: > .smm. in either group blood transfusion results in zt significant increase in hb (a: . _+ . to . + . g/dl; b: .(~ . tt, . + . g/dl; i: . -+ . to . -+ . jdl; i : . -+ . to . + . g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: -+ to -+ ; b: + to + ; : -+ to -+ ; i : -+ to -+ ), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i -+ to -+ ; b: -+ to -+ ; i: -+ tu -+ ; : -+ to +_ ); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of > . -+ . g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in . % of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a -years-old worn an with a breast cancer, diagnosed in , initial staging t nim , treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of mg/m until august . the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of mg paclitaxel, given over hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to %. she died days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selectin (selam- ). sicam- , svcam- , selam- , and inflammatory cytokines were determined. methods: sicam- , svcam- , and selam- were determined by elisa. tnf-a, il- , and il- were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam- and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam- level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il- , but the level of il- . discussion and conclusion: the slcam- and svcam- levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il- also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam- , elam- , and vcam- , appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut- , reduced release of inflammatory cytokines (tnf, il-lg, il- ), activation of complement (c a, c a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut- on soluble adhesion molecules following cpb surgery. patients undergoing cpb surgery were divided into two groups, group a consisted of patients who received omg of fut- in priming solution, followed by a continuous infusion at mg/kg/hr during cpb in addition to initial heparin dose of mg/kg. group b, a control group, included patients who were injected with heparin only. the plasma slcam- , selam- , and svcam- concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam- and slcam- reached their peaks on hours after cpb and on pod respectively in both groups, but they remained lower in group a (selam-i: . + . vs. . • ng/ml, p< . , slcam-i: • vs. • ng/ml, p< . ), svcam- , in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut- reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, l- , il- , il- , s-cd- and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: - ml/h, filtration rate ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock ( ), the so called hepatorenal s~aldrome ( ) and a severe pancreatitis ( ). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha ( cases), scd ( cases), il- (l case) and il- ( cases), hemofiltrates contained no activities. only il- was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il- and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il- , il- could not be eliminated in cvvh with a filtration rate to ml/h. in contrast to findings of other investigators with a higher filtration rate (> ml/h), we found no significant concentrations of tnf-alpha and il in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (> ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~ ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed times using nafamostat mesilate as an anticoagulant in hours with l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~ -proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = consecutive pts treated with hd; n = consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = _+ ; mean number of organ system failure (osf) = . -+ ; mean blood pressure (mbp) = • mmhg (first day of application of hd). cvvhf-group : apachen score : + ; osf = -+ ; mbp = + mmhg (first day of application of cwhf discussion : during the first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) patients with mof were treated with cvvh in the period between january in to march in . the mean (• age of our patient population was , • years, being % male and % female the whole patient population was with mof iust at the moment the technique was accomplished; % was in mechanical ventilation, % needed vasopressor support and % required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was , ~: , (range - ) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine , st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta and dopaminergic receptor agonist. the specific beta adrenoceptor antagonist ici has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs ( - kg) divided into groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and pg/kg ici bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at - mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b antagonist blocked any protective effect of endogenous beta adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr chloride, a beta and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta adrenoceptor antagonist ici was infused to block any protective effect of dopexamine. nine anaesthetised pigs ( - kg) were randomised into groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition pg/kg ici bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at - mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. . _+ . , dopexamine . + . ", blocker . + . . data expressed as mean + sd. significance p< . . * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) ( patients) with bilirubin > . mg/dl and lung injury score < . , it) mof-ards ( patients) with respective values < . and > , iii) patients with head injury with respective values < and < , served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index > ml/min/m prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was . _+ . vs . _+ . (ns) and . _+ . respectively, of the paop was . +_ . vs +: (ns) and . + . respectively and of the cvp was .+. . vs . + . (ns) and . respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was . + . vs . + . ( < . ) and . _+ . respectively and lung injury score was . objectives: oxygen delivery (do ) and oxygen consumption (vo ) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo determination by either indirect calorimetry (vo m) or tick calculation (vo c). the purpose of this study was to investigate the changes in vo m and vo c following do increase. methods: the relatives of critically ill patients (mean age years, mean apache ii , mean mof-score ) gave their written informed consent to participate in this institutionally approved, prospective study. do was increased by fluid loading (hydroxyethylstarch %: mean volmne ml, mean duration of infusion min) and catecholamine support (dobutamine: mean dose , ~g/kg/min). changes in vo m and v c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor integrated in the ventilator (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in patients with peritonitis of different origins ( nonsurvivors and survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol ( +_ , nmol/ i), aldosterone ( , • , nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=- , , p < , between cortisol and pao ; r = , , p < , between cortisol and d (a-v) ; olso renin -cao r=- , , p < , , renin d ~,vl o r = , , p < , ). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b and p fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next hrs fzom + ml/ hrs to + ml/ hrs (ns). data expres,uxl as mean + sem. three patients ( %) became polyuric with urine output > ml/hr within days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was . + . days. serum potassium levels were well controlled. day or immediate pre-dialysis levels were . + . mmol/l compared with pre-lreatment . + . mmol/l overall mortality in this series was / ( %). duration of acute dialysis in survivors with renal recovery was . +_ . days. patients ( %) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do and vo calculations and temperature) and determined the respiratory mechanics (compliance and pao /fie relatinship) before starting the procedure, after minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler and minutes of zero fluid balance bemofiltration and after minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for minutes without filtered fluid production and after minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate ( mosm/l), was injected in the dose of .o ml/ kg into v. cephalica antebrachii after hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco and phi obtained simultaneously by gastric tonometry. material and method : in a patient ( y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : sets of data were obtained and are expressed in mean + sd. gastric pco z was , + compared to , + . mmhg for portal pco . phi was . +._ , vs . +._o, for portal ph. no correlation was found for these parameters. p (gast-a) c was . + mm hg vs + . mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = , ) [figure] but no correlation with p (gast-a) c . obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange ( ). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator dilution method, with indocyanine green and the stable isotope h as tracers, was used to measure evlw ( ). arterial bloodgases and endtidal co were registered. evlw data was compared to a group of renal healthy patients ( ). dcp n evlw, ml -pao , mmhg h~o +, nmol/l control group - -- l _+ "* -+ _+ crfgroup ii -+ ~ +- ns -+ "(" iii +- t _+ ns -+ t ** p < . dcp i from dcp , t p < . dcp li or i from dcp i, :~ p < . dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco ( . + . kpa) and etco ( . + . kpa) were unchanged while h o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao . the reduction in h + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao in bicarbonate dialysis. . prezant lung ; : - . . wallin j appl physio ; : - . a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value . , can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h -bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below . . results: among the patients, had a drop of phi below . during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi= . and the second . . the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the th day after operation and died on the th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other patients with a fall of phi had a lowest phi= . . . . . . . respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the th pest-operative day and a dehiscence of the surgical wound on the th day: the third one, operated on for a sigmoid carcinoma, had melena in h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these patients were discharged alive from the hospital. the other patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of hours duraticn. all l:atients nm.'-~ms_(~lly va~ ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e rain after the ~, ~ne at the middle, ~ne rain before lhe erd ard one rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re ( ve)emm to . ~ ~, ) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n to ~mg hg, ) i~ from to . mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o. ) of: i) s~o~ from to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: patients ( males and female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. patients were operated on eleetively, had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a month period ( / - / ), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to , deliveries occurring during that time period for a . % rate. the following procedural indications for icu transfer occured: insertions of endotracheal tubes, arterial catheterizations, temporary tracheostumy, and venous catheter. the following medical diagnoses were found: hypertensive/eclamptic episodes, hypotensive episodes, pulmonary embolism, mitral stenosis, and coagulation deficiency. there were four episodes of respiratory failure ( %). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for months in a multidisciplinary -bed icu. methods: data from consecutive patients admitted to the icu from / / to / / were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm , mpm and odin. data from days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = %) were evaluated from data collected during the day of admission (saps ii, rpm , odin) or after hrs (mpm ). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary -bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for consecutive patients admitted to the icu from / / to / / . data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied men and women, with a mean age of years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from clinical variables and laboratory items for patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i . thresholds were calculated with the youden's test (se+sp- ). results : patients were admitted in the intensive care unit (icu)and patients in medical units; patients died. the saps ii was higher in patients which died than the survivors ( , + , vs. , + , , p< , ; respectively), the best threshold was , se was de %, sp was %, the ac was / . thus for patients admitted in icu than in medical unit ( , : : , vs. , + , , p< , ; respectively); the best threshold was , se was %, the sp was %, the ac was %. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, hiv positive and hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure ( % versus %, p < . ) and septic shock ( % versus %, p , : ) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. , n= ) and january-march (period , n= ) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache scores. the icu and hospital mortalities decreased from . % to . % (p=ns) and . % and . % (p=ns) respectively in period . the mean icu and hospital stay was also decreased from . __+ . to . + . days (p=ns) and . __+ to . • . days (p=ns) respectively. the improved micu length of stay for survivors in period was statistically significant ( . • . days vs . • . days = . ). there was no significant differences in the vse of peritoneal dialysis ( . % vs . %) and mechanical ventilation ( . % vs . %). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from % in both to . % and . % respectively in period . conclusion: the duration of critical illness was reduced in period . hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, ald patients ( males; females; median age years, median apache ii score ; hospital mortality %)with severe complications ( [ %] with bleeding oesophageal varices, [ %] with hepatorenel failure, [ %] with respiratory failure, [ %] with septic shock, and [ %] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians and , p< . ), or more organ systems in failure in the first hours of icu admission was associated with a % ( ) mortality. mortality was also related to child's grading and the numbers of organs supported. of the patients who received more than one form of organ support only one survived. organs supported mortality a (n= ) % none / ; % b (n= ) . % one / ; % c (n= ) . % two or more / ; % the icu costs for the patients were only s but s ( %, patient days) was utilised by the patients who died, giving an effective cost per survivor (ecps) of s . although the cost of treating the patients with or more organ systems in failure on the day of admission was s ( % of the budget) the ecps was s . conclusion patients with ald who have or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which surgical patients were randomly assigned to either a control group (n = to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = ) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at / prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality days postoperatively. results: in the protocol group, there was a % reduction in mortality days postoperatively (p = . ), a significant reduction in the number of complications (p = . ) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a . % survival rate days postoperatively was s , , resulting in an average cost of s , per surviving protocol patient. the average cost of a control patient was s , to achieve a . % survival rate days postoperatively, resulting in an average cost of s , per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s , per patient together with a % reduction in mortality at days postoperatively. hence, the cost per surviving protocol patient was s . less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet ; : - ; madl et al. lancet ; : - ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: recordings of standard bilateral sensory evoked potentials were performed in patients with acute liver failure. findings were focused on cortical n peak -a stable negative deflection occuring • ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, patients were referred to emergency liver transplantation, and patients died. in all patients who recovered spontaneously, n peak was detectable between and ms. all patients who were referred to liver transplantation and of patients who died, developed a loss of a prior detectable n peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \ -test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows . . was used for statistical calculations. results: in-unit mortality: / patients ( . %) --oth . % > as . %> ni . % > ed . %; p = . . in-hospital mortality: / patients ( . %) --oth . % > ni . % > as . % > ed . %; p = , . mean survival time in days after discharge: as < ni < oth < ed ; p = . . conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january asd ]lay data from , patients over years of aqe who retained in the icu for mare than hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s . % and in-hospitul mortality . %. patients who died showed a higher spas score then survivors ( , ~ i ,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection ( ~ . ; % el, . to . ), pmo~othor~x related to mocasnical ventilation (@ . ; % cl, . to . ) and delay in the insertion of a fln~-quidod catheter (ii~ . ; % ic, i.i to . ). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~ , j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~ i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january and april , all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of intnmtsd patients amre stndied. self-extu~atien occurred in ( . %) patients and . % required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe ( . v_s . years, p = .~ ), ~verity of illness ( . ~ . spas score, p = . ), dia~isstia category ( s. % v_s . % of patients with res~iratury conditiono, p = , } and mean length of stay ( , ~ , days~ p = . ) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a . iir for mortality ( % ci, .i to . ) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber all patients who fulfilled criteria to be almittod to the ic who for waste~r reason retained in tile observation unit for more than hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of , patients who suffered no delay is admission to icii over a period of years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~ &ults: a total of patients remained in the observation nit for more than hours with a del w in igd admission of . _+ . hoers. assisted mechanical ventilation was requited in % of patients and only monitericatien in %. itsse patients were cspared with ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn % as compared with . % for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = . ; % ci, , to , ). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was for survivors and t for non-survivors (p < . ). the mortality ratio was . and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent ( ) of icu bed days were occupied by % of patients (non-sarvivors) which resulted in cancellation of cardiac sot#cat sessions in momhs. conclusions: this study concludes that apache t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a day period. ) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december through to april , patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score= , maximum day off scorn= , sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at . (two-sided), with n/group = [(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as to patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from % to % ( % relative risk reduction) where the n/group= . scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. . non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" . autonomy: in every particular medical attendance, the patient has ability to decide by himself. . justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. ( - ) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p = . ). the mean aiii score when considering all admissions was , ( - ) . the initial score between survivors and non survivors showed ststistical difference ( . vs . ) respectively (p < . ). univariate logistic regresion analysis demostrated a % increment in death probability for every points augmentation in the aiii score with a sensitlbity of . % and specificity of . %, the roc curve showed that the best cut off point for death prediction was points with a sensitivity of . % and specificity of . %. if a patient is classified as high risk (> ) the bayesian analysis showed a . probability of death and for one class(fed as low risk (< ) a death probability < %. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama , : ] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. , : - ] and then a step verbal scale (pqolv): best, good, fair, poor, worst. of the studied patients, at the first interview, were able to use both scales, but ( . %) understood only the verbal one. at the second interview, patients were not able to answer, used both scales and only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, pts ( . %) died in icu. they were males ( . %) and females ( . %). their mean age was (+ ) years and mean ef was . (+ . ). nineteen pts ( %) had low (< . ) preoperative ef. mortality was . % in the coronary artery bypass grafting (cabg) group (n= ) and . % in the valve replacement (vr) group (n= ). in the cabg +vr group, mortality was . % (n= ), and . % in the remaining pts (n= ). cardiogenic shock was the sole cause of death in pts ( %), septic shock in pts, whereas sepsis in combination with ards in pts, sepsis and stroke in two pts. in addition, pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p< . ) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: . although cardiogenic shock is the main cause of death ( %)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. . patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. . although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after and up to hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from / to / consecutive pts with ami were treated by primary ptca in the wuppertal heart center pts ( , %) were admitted to our hospital > hours and < hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was years ( - ). pts were male, four female. % had an anterior wall myocardial infarction, % suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in . %, multi-vessel-disease in . %. average time of onset of pain to recanalisation was min ( - ). angiography revealed timi-flow in . % of the pts, timi-flow i in . %, timi-flow ii in . %. average follow-up (fu) period was months ( - months). timi iii lv-ef ~ -day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality . % %/ % . % . % . % % early mortality occured in the two pts, who were in cardiogenic shock at admission no pt required emergency coronary artery bypass grafting.restenosis > % was seen in % of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about % (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of icu's representative of the italian icu system methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the patients recruited during one month period, a total of patients were included in this study. for the purpose of the comparison of the two scores, patients with less than years, or having cardiac surgery or staying in the icu less than hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$ ~: saps ii fits the data equally well compared to the older version (goodness-of-fit p= . and in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve . and . , respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p-- . ) and discrimination (receiver operating characteristics area= . ) were observed. c nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: ) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); ) assess the efficiency by means of a resource use index (rui); ) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. critical from ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was . and it ranged from . to . in the icus. the overall rui was and it ranged l~ont . to . . there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine ; : - ): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on random admissions to specialty icus with beds (range - ) between february i and december l, . post-operative solid organ transplant recipients were excluded. units included general medical, general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from . - . ; predicted risk of hospital death ranged from . - . %. standardized mortality ratios ranged from . to . with icus performing significantly better and performing worse than predicted (p< , ). los ratios and icu readmission rates ranged from . to . (ns) and . to . % respectively. patients predicted at low risk of requiring active icu treatment ranged from , to . % conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals ( - ; - ; - ; - ; > ) , we compared tcu mortality within age intervals (< ; - ; - ; - ; - ; > years - , - , - ) . in these groups mortality may be twice higher in the > years patients than in the _< years. mortality does not vary with age in low (physio a n = - ) and high (physio a n = > ) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear ( ), patients were treated at the unit, of them died, and ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only patients were evaluated on the fu. the, were classified into the follov ng three groups: acute medical, elective surge d and acute and emergency postoperative. the patients were seen at , and months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~ ous jobs. apache scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that / patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and % showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only % had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii i mpmp ii ), apach ii end gaprik were evaluated o~ patients with acute myocardial infurction admitted to intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii , saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii , and apache-saps were adapted and reduced to (mpm ii cor) end to variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to or variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included consecutive intensive therapy unit (itu) admissions with an itu stay> hrs in a month prospective study (exclusion criteria: burn injury and age < yrs). data were couectsd applying the criteria described by the developers [ , ] . the definition of coma (mpm ii) was modified and the best assessment within in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was yrs (ranse: - ) with a male:female ratio of . : . the actual hospital mortality was . %, mean predicted death rates were . % (mpmz ii) and . % (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p< . l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion %: apache ]/i . %, mpm ii . %). the area under the roc curve was . (ap iii) and . (mpm ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~ - , mpm b ;( - , lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -< % with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks > % ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of adult patients consecutively admitted to a medical-surgical itu over a period of months. patients with burn injury, age < yrs and itu stay < hrs were excluded. using a eomputerlsed database, we routinely recorded hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;( ) statistics and calibration curves [ ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the male and female patients was yrs (range: - yrs). of these patients, % were medical admissions, % were admired after emergency and % after elective surgery. the observed hospital mortality was . %, the overall mean predicted death rate was . %. mean predicted risks were siguifieanfiy greater for nonsurvivors ( . %o) than for survivors ( . %, p< . l, t-test). apache iii showed good calibration (z -~ , lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was . % (decision criterion: %). the area under the roc curve was . % confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected patients, in two months in summer in all over italy. regional data were echieved in a three years entlection ( -i ) in lombardia' icus from archidia group (arehivio diagnostieo), including patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr observed patients account for . % and % of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data ( . %) and extremely low mortality in regional data ( . %). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: . % and . %; .a.p.s. score: . and ; iength of stay: % and ). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively % vs. %, medical patients were % vs. % and traumatised were % vs. %. % of "observed patients" in national study and % in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than % did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients ( % vs. %); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during . the authors collected data of patients outcome and predictor variables. overall mortality rate was , %. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death ( %). average length of stay was . days ~. % patients died in the first ten hosiptal days and only % after days. age was directly correlated with death % of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose ( %) and natrium ( %) were in optimal physiological values. serum proteins ( %) and haemoglobin ( %) levels were inversely related to death. multivariate showed that alveolo-arterio difference in content was the most informative of all mortality predictors (mean value , mmhg in % patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. ; : - ) and the sepsis score of elebute and stoner (br. h surg. ; : - ) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past years for patients in our icu was calculated = % (prior probability). the outcome of patients who were admitted to our icu for > hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died ( . i . v~s . • . and . • v's . • . respectively p < . ). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at , and h while in the intervention group phi measurements were made hourly for h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure > mmhg, systolic arterial pressure > mmhg, urine output > . /ml/kg, haemoglobin > g/dl, blood glucose < mmol/ , arterial oxygen saturation > % and correction of uncompensated respiratory acidosis. if the phi was < . after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at then mcg/kg/h, titrated against phi. this additional therapy was continued until h after entry into the study. in each year patients were subdivided in two series with random selection, so that the st series contained abeat / and the nd / of the patients. the st series of all the years constituted the devdoping data set and the nd series the validation data set. with data of the st series ( patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die st, th, th and th day, calculating for each lime the apache ii score (for a total of records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january and february , anonymous questionnaires were sent to intensive care units, all over greece. results : questionnaires ( , %) were replied and returned back. of them , % were answered by male and , % by female. the doctors replied in the following rate : , % aged up to , % aged between and , % aged over . questions were answered and were divided into main topics, as following: . admission criteria: limited bed availability was the main cause for refusing admission in , % of icu's. , % evaluated each case's viability and only , % used some prognostic score system. , % of icu's accepted all cases and a significant percentage ( %) gave in to pressure coming from their colleagues ( , % female and , % male). . informing the patient/relatives: only , % was willing to tell the whole truth, while , % had given selective information.. in the case of iatrogenic incident, , % withheld it, because either they feared legal implications ( , %), or lost of trust ( , %). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of , %, while only , % found informed consent necessary for the proposed treatment procedure. . withdrawal of therapy/dnr orders/organ donation: , % were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in , % such a decis~n was unanimous, while the percentage of those carrying it out was , % ( , % female, , % male). in case of brain stem death , % ( , % female, , % male) withdrew any life support. , % would like therapy withdrawal to be legally established, while only , % would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only , %. , % considered organ donation to be a necessary proposal, while , % refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it ( , %), or because they doubted the procedures' objectivity ( , %). note: in greece, icu beds are less than % from the total number of hospital beds available. only a percentage of - % of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n= ) and mitral valve replacement (mvr) (n=t ). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at hour and at hours after operation. results: there were no significant hemodynamic changes during the first hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t trauma and sepsis patients randomly received either . mg/h (group i, n= ) or . mg/h (group , n= ) of enalaprilat i.v. or saline solution (control, n= ) as placebo for days. plasma levels of endothelin- (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion ( . mg/h: from . • to . • pg/ml; . mg/h: . • to . • whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p< . ) and decreased after . mg/h of enalaprilat. et remained almostunchanged in group , whereas et increased significantly in the control patients (from . • to .t• on the th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p< . ), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, min after the patient was placed in a trp and again min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo by monitoring partial concentration of o and co ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco without a similar trend in vo values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after min in trp. evaluation of vo from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after rain. since vo did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo ximetrix, abbott) allowing continuous svo monitoring was sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for hours and/or successful weaning (s-w). (j physiol ; ." - ) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and % co inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, year, performed three incremental exercise tests in a randomized order: ) inspiring air without vd (air control, ac); ) inspiring air with vd of ml (avd); ) inspiring % co ; % oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to % peak hr. results: ventilation (vi) was significantly greater (p< . ) during the avd and co tests than during the ac test at the same work rates. end-tidal co (petco ) and estimated arterial co (paco ) were significantly greater (p< . ) at w and w. oxygen saturation was significantly lower (p< . ) during the avd test than during the ac and % co exerdse. at matched hrequivalent to % peak hr, vi was significantly greater (p< . ) during the avd and % co tests than during the ac exerdse ( l, l, and /). conclusion: we conclude that the increase in xri and petco due to vd loading and % co inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu ( men and women; mean age . - . years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was . - . hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt ( , , and hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (< hours). table i list the variation of plasma eoneenlrations of tbars (mean -sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe time cuncentzatiens reveal a difference p<o. between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~). ) at , and hours fi-om de begin~g of'it. comparison ofthe , and hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in healthy subjects (group i) , patients with several risk factors of ischaemic cardiopathy (group if) and icu patients with possible aim ( were confirmed ami (group ma) and were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i . electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h , s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< . ) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i ( pts) -p:ithout d and group ii ( pts) -d ~as administered . mg/kg iv bolus hours prior to anesthesia and . mg/kg/h upon the end of operation and for at least hours post-operatively. iv was substituted by oval administration of x mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during minutes starting minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of o fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period ( hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), % survived, the remainder died of incomplete recovery. in vi with bvad, only % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in patients by comparing paopee, paoptmc, and paopnadir obtained within the first seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in patients (group a) but present in others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : +_ ; gr b : _+ cmh ). results : l. gr a : paopee ( _+ mmhg) differed (p < - ) from paopnadir ( • mmhg) and paoptmc ( _+ mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves ( " - , + , + mmhg respectively). . good correlatkm (r - (i. ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). . lung compliance (v t/bpah,) correlated well with i t in both groups (r - . ), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (> ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) hours after operation, e) hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline hours after operation. methods: patients with preoperative lvef< % undergoing coronary artery bypass grafting were studied. after surgery, a f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z- t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m ), intrathoracic bood volume (itbv,ml/m ), pulmonary blood volume (pbv,ml/m ) and extravascular lung water (evlw,ml/kg). with a f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while extraction ratio (o exr,%) and oxygen delivery (do ,ml/min/m ) was calculed. peak inspiratory pressure (pawp,cmh ) and mean airway pressure (mawp,cmh ) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after minutes (to) of volume controlled standard ratio ventilation (vc), and after minutes (ti) of stabilisation period of pcirv ( % inspiratory time, % pause). vt,ve and total peep were held constant in every mode of ventilation. +_ . " *'p < , versus to conclusions: these data show that pcirv : is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo . paw eight patients ( melee end females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so /co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o. ) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do and tissue oxygenation (pro ) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do and pro . objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight + kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio . ). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao ) and mixed venous (pvo ) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, pm microspheres (sephadex g coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to mmhg initially. rain after embolisation when haemodynamics were in a steady state (control ), inhaled no was administered. further measurements were taken min after ppm no (no ), min after ppm no (no ), and min after discontinuation of no administration (control ). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given rag of actij.yse as ~ hrs infusion, followed by heparin~single j. ihjaction snd iefusion ie doses - j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im . . hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao and sao in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed % improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation ( ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv ( ). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: normothermic who were stable (< % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit to insure both normal abg and a peak airway pressure < cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi. , ) with synchfjv runs interposed (hfjvz ). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > % ; n= and < %; n= ) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate h-bpap. trace mnou~ of ~fi-bpap ( gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of . pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g -a: %, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to • reflecting a • inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to :l: %, representing a -a: % inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as . x " sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi ) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from + to -+ dyn*s*cm (p< . ) and pulmonary artery pressure from . + . to + . mmhg (p < . ), cardiac output increased from . + . to . _+ . i/rain (p < . ), mixed venous oxygen saturation from . _+ . to . + . % (p < . ) and arterial oxygen saturation from . + . to . _+ . % (mean _+ sem of trials in patients). -month iloprost nebulisation in one patient ( gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selecfin (selam- ) levels in patients with acute myocardial infarction admitted within hours after onset. peripheral venous plasma-samples were collected at the time of admission, , , , , and hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into groups as follows: group ; killip's class (k) and without thrombolytie therapy, group ; k and with thrombolytic therapy and group ; k and . both plasma sicam- and svcam- concentrations in group and were elevated rapidly and significantly and maintained at a high level during the first days. plasma selam- level did not change in any of the groups. these results suggest that the adhesion molecules icam- and vcam- may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no no -(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were patients admitted within hours after onset. venous blood samples were collected at -hour intervals on the first day, -bour intervals on the nd day and -hour intervals on the rd day and th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about hours and - days after onset (basal level; . _+ . , first peak; . !-_ . , second peak; . + . ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of hours after onset were significantly correlated with maximal plasma creatine kinase level (r= . , p< . ). the levels of plasma nox in the early stage of myocardial infarction (from admission to the th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range - ) fullfilling the high risk criteria of shoemaker (colectomy , gastrectomy , pancreaticoduodenectomy , others ). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci > . l/min/m , pcwp > mmhg, hb > g/l, sat >. . objectives: evaluate the acute effects of , mg ipratropium bromide and , mg fenoterol (ibf) inhaled dose on pulmonary function in nonsmocers (nb:m) and smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and healthy subjects. methods: pulmonary function tests were performed < h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred puffs-ipratropium bromide ( , rag) and fenoterol ( , rag). for , h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in second(eevt) was , % (p< , t) improvement and , % (p< ,ob), forced expiratory flow betwen % and % of the forced vital capacity (fef . ) was , % (p< , ) and , % (p< , ) and maxamal voluntary ventilation (mw) was , % (p< , ) and , % (p<o, ) improvement. the mean changes in normal subjects were mild (fev increased by , %,fef - by , % and mvv by , %). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group (n= ) received both imas as grafts and group (n= ) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao /fio ratio was measured in all patients in the icu, at , , minutes on mechanical ventilation (mv), at , , minutes after extubation (ex) and on postoperative day (pod) . in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group had significantly longer bypass ( _+ vs + . p< , ) and aortic cross clamp time ( +- vs + , p= , ). pao /fi ratio was significantly higher in group only at minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group ( +_ vs _+ , p= , ), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group and one case of pulmonary infection in each group. after extubation m[n min min min* rain min pod ' _+ _+ + -+ - -+ _+ _+ _+ _+ -+ _+ _+ -+ mean_+sd, *p=o, o conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in patients without previous pulmonary disease undergoing elective cardiac surgery. there were males and females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were _. and • min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb- h after h after stemotomy closed chest cpb cpb estrs (cmh /l) . + . . - . " . _+ . * . - . * est cw " . _+ . . • . + . . -+ . est l " . -- . . - . " . -_ . " . _+ . " x + sd. * p < . : anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first hours and stabilizes hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied pts, % ( / ) developed dcm during the follow-up study. among this group % ( / pts) were in class i and ii and % ( / pts) in class iii and iv of the nyha classification. we divided this population in a acei+ ( pts) and acei-( pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t and t type cell population and its t /t ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography ( -de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new -de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of idcm pts, mean age _+ yrs, % male gender, using a combined -de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated -de software. computer processing and analysis of the tee images led to a -de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using -de derived indices. mean values of -de lvesv= -+ ml, lvedv= _+ ml, lvstv= _+ ml and lvef= -+ % were obtained. for all these -de parameters, good correlations were observed with -de lv measurements obtained through biplane tee analysis of the lv cavity (r>. ; p<. ) as well as regional analysis of sequential -de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new -de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of dcm patients, mean age _+ yrs, % male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ % pts; group slvd- % pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. -de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. + -_ . simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with (fl ) and (fl ) ml of dextl'an (rhemacrodex| in patients free of previous cardiopulmonary disease ( • yrs) with acf (ci < . l/rain/m ) due to angiographicalty proven mpe (miller score > ) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = . ) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep ( - - - cmh ) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in mechanically ventilated arf patients (lis = . ~- . sd). according to the response to peep , two groups of patients were defined: group a ( pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep , rvef decreased at peep (~ . %)~ rvesvi increased only at peep (+ . %) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep , 'rvesvi decreased only at peep (- . %), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the levels& peep were studied. rvedvi and ci were significantly correlated in out of:l patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in out of patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in patients of group b and in patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is . + . nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only • . nun hg is tolerated (p < . ). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at , and minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after minutes of reperfusion there was a highly significant increase (p < . ) in the mda values (mean = . /zmols/l). these returned to basal levels after and minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used "large white" pigs. mean weight was kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends patients studied as two groups of individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered rag/ hours of vitamin e (tocopherol acetate) hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a -year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of . u/hr for hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed ( . ui of streptokinase on days , , , ). fluid drainage was improved and cardiac output was also improved (day : . .min "i, day : . l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload ( , ) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of successive breaths of increasing magnitude ( , , , and ml/kg). a line of best fit is drawn between the minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh ). in mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of ml/kg of plasma expander. the initial mean downslope of the rsvt was -. + . mmhg/cmh . following volume loading the downslope decreased to -. + . (ns). at the same time, cardiac output (co) increased by . + . l/min (p<. ), end-diastolic area (determined by tee) increased from . + . to . + . cm (ns), and paop increased from + to + mmhg ( p < . ). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = . ) and the eda (r = . ). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion ~ . g/kg/ rain with a map --< mmhg. cardiovascular support was limited to na + dobutamine (db). c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t - ); and at the end of treatment (t - ) with c . conclusions: c does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to h. c is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope hzo in aorta (q v~ o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h ( . ml), h ( . ml) and icg ( mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and h in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= boluses) over group i (n= boluses) ( . <- . vs. . +- . , p= . ). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz ( ) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope h for the estimation of extravascular lung water (evlw hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg ( rag) and h ( . ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q n o, t o) ( ). data analysis: evlwg~av was reference for evlwzhzo calculated as q hzo times the difference in mean transit time between t nzo and rico (atm n). as reference for atzn o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for h was calculated as the sum of central blood volume and evlwg=v. boluses were administrated in a group (i) of anaesthetized pulmonary healthy sheep while q was altered. another boluses were administrated in a group (ii) of anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. results: for boluses h parameters were not significantly different from their respective reference parameter: at vao . +_ . s vs. atg~, . + . s, evlwzh o -+ ml vs. evlwg~,~ + ml. in group i the ratio between hzo parameters and respective reference parameters (n= ) were independent of qlco from . to . l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg ( mg) in water ( ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( men, women ) age from to were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in % ( pts), without high pvr-in % opts) and with left ventricular failure-in % ( pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed- ~-pat~e~ts ( males and woman), average , years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture ( ~ ~ ~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from . +__ . mm to . +_ . ram (p = . ) at ~ tilt angle, and from . + . mm to . + . mm (p = . ) at " tilt angle. the tidal volume increased from + to + rut a * tilt angle, and from + to + ml at * tilt angle (p = . ). two patients could not bear the horizontal position ( ' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion ( . + . mm to . + . ram). after an overnight respiratory assistance, pao increased from . +_. . to + . mmhg ( = . ), sao increased from . + . % to . +_. % (p = . ), and paco decreased from + . to . +_. mmhg (p = . ) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity and l/min) vs pressure triggering (-lcmh ) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized min. runs using a bird st ventilator at zero peep (zeep). trigger values for pressure (-lcmh ) and flow ( l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv ( . + . cmh: x sec) and a/c ( . + . ) with pressure triggering, as respect to psv ( . + . , p< . ) and a/c ( . + . , p< . ) with flow triggering ( l!m). no differences were observed between and l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being . + . cmh (psv flow trigger) vs . + . (psv pressure trigger) and . +_ . (a/c flow trigger) vs'f~ +l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung . we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in anaesthetized and paralyzed piglets we applied short periods ( s) of one-sided ventilation ( breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied -to-r and r-to-i cross-talk at ventilatory rates of , and bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during bpm the r-to-i crosstalk was _+ . % (mean +__ sd) of the tidal volume to the right lung and the -to-r crosstalk _ . % of the left tidal volume. both values increased at bpm to _ . % (p < . ) and _ . % (p < . ) respectively. the values at bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = ) or as a slow infusion (n = ) in infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every minutes with a lung function cart (sensormedics ) without interrupting ventilation. infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > %, maximum r > % of baseline value. retreatment was followed by an increase in r in all patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores > ) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l. and a peep level < cme=o} was not able to get either a p.ojf=o= ratio > or a s.o= > %. patients were sedated, paralyzed, and a ventilator (serve c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at . and peep removed. continuous gas flow ( • ml/kg] was humidified and jet delivered through a tube ( ram id, ml capacity, . ml/cm h=o compllancel ended in a nozzle ( . mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo ) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin . further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies - hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies - hz with the relative inspiratory time (ti) . - . . the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats ( - g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature ~ the arterial pressure was monitored. after traeheotomy the metal cannula ( mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = . kpa, ti = . . the dead space of ventilator including canula was . ml. the initial frequency was hz and rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : hz--> hz--> hz--> hz--> hz--> hz--~ hz--> hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco = + mm hg. the arterial po was always above mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt = . --+ . ml/kg for ft = hz, vt = . + . mukg for fz = hz, vt = . +_ . ml/kg forf = hz, vm = . + . ml/kg forf = hz andvmt= . + . mukg for fs = hz (presented as mean values _+ s.d., n = ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = . * f-e. . conclusions: the described system allowing ventilation in a wide frequency range - hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr - and gacr nr . s intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on ventilated patients (puritan bennett ac with metabolic monitor pb set to measure end tidal co ). all measurements were repeated with the patient stabilised at cm. cm and cm peep. inclusion criteria were: ) haemedynamic stab(l( .ty for hr; ) pulmonad" anon" flotation catheter in situ: ) volume control ventilation with plateau of . s: ) fio ~ > . to maintain pao~. > kpa with em peep: ) qs/ot > %; ) pao /fio ratio < . measured variab!es included: r minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co ; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p< . ), compliance (p<t). ) and o,'gcn deliver, index (do , p< . ) occurred with increasing peep (table ) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of men and women were studied (n= ). the mean distance of ett tip to carina was . cm. using the reference markings, cases ( . %) had the ett tip < cm from the carina and cases ( . %) > cm. one case resulted in an endobronchial intubation. the mean height of all patients were cm ( - ) for males and cm ( - ) for females. of the patients with ett tip < cm from carina, the mean height was cm and cm respectively. ~ onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases ( . %). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp- pulmonary monitor were recorded hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre . _+ . to post . _+ . , p < . ). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group included eight patients who were completely weaned from ventilator; group included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups ( . _+ . vs . + . in peepi; . _+ . vs . _+ . in cld, p < . ). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group ( % % in group vs . % in group ). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were survivors and deaths in this series (mortality %).using arterialpco values measured hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was %. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was %. arterial co accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po , pc ) and the ph (abg) was examined in stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for and ps plus intermitted mantatory ventilation in ii. [n each patient, speciments of abg were measured at min intervals during a - study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was . percent for po , . percent for pco and . percent for hco . the average sd for ph was . , the corresponding c for systolic bp, diastolic bp, cf, tv, rf were . , . , . , . , . percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in ; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, del, vo by improving the rheologic properties of blood and plasma, normalizes ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by . - . limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo ) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in critically ill patients treated with mechanical ventilation for at least h, who fulfilled at least of standard weaning criteria (vt> ml/kg; respiratory frecuency (f) < ; pimax > cm h ; pao /fio > ). baseline measurements: t, vt, p . , pimax, f/vt, p . *(f/vt), p . /pimax. study protocol: measurement of vo , vco (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after and minutes of spontaneous ventilation with cpap cm h . the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, were extubatad, and of them had to be reintubated before h, being considered also weaning failures. during cmv, vo /kg was . + . ml/kg/min, and . _+ . mlo- /kg/min after ' on cpap cm h (p < , ). of patients ( %) with standard criteria were extubated, while only of ( %) with criteria (p< , ). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa ( . - . ml/kg) in anesthetized, paralyzed, and intubated dogs (n= ) during volume controlled ventilation: rate= /min, peep= cmh , ti/ttot= . , fio = . , vt= ml/kg. animals were rotated during the oa infusion and the following minute stabilization period to assure uniform injury. in the supine position, peep was set - cmh above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of cmh : animals were ventilated in either the prone (n= ) or supine (n= ) position for four hours. pulmonary artery occlusion pressure was maintained constant ( - mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale - ). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at ( to ( cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two rain periods of chfo (sequences : ( and ) c/rain : group l, n = l: and c/rain : group , n = ). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa is not significantly modified during chfo at or c/rain. paco is slightly decreased at c/rain (p = .( ). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. ). there is no more effect of chfo for patieets who are at a flu higher than . (n = ). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < .ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = ): two levels of assist were studied; flow assist (fa) of cmh /i/sec and volume assist (va) of cmh /i (assist ), and fa of cmh /i/sec and va of cmh /i (assist ). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist ) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco of mmhg v t was increased significantly by . + . i and . + . i in protocol a with assist end , respectively, and by . _+ . i in protocol b with assist (p< . ). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ ( mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco ( mmhg), pdi and pmus at the end of neural inspiration decreased by . -+ . % and . + . %, respectively, from control values. neither change was significant (p> . ). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol ) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps ( - breaths each) from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p< . ); at i/min ttot, expressed as % of that at i/rain, was . +_ . % during sleep and . +_ . % during wakefulness. during wakefulness, at i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was % of that at i/min (p< . ). the corresponding value during sleep, was % (p> . ). in four sleeping subjects the increase in v~ was sustained for . - min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only - breaths. we conclude that ) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and ) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively asa i pediatrics patients ( to yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of mm. id and . m. long (children < kg), and a corrugated dar set of mm. id and . m. long (children > kg) were respectively used for ccl an cc values of . and . ml/cm h . a vtof mlg/kg and respiratory frequency was adjusted for an end-tidal co (etpco ) between mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was . % and reached as high as . %. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as . %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago- ) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically patients. the ventilatory setting is pressure support mode. the pressure level and fit were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: '(immediately before h/d), ', ', ', ', ', '. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp- ). the mean value of the breaths minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po and pco> however, a potential problem in mechanically ventilated patients is that the l mixing box (mb- l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb- l on the v c were compared with those produced by a new l mixing box (mb- l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb- l was substantially higher (vc= . *ppeak) than that provoked by the new mb- l (vc= . *ppeak). at a ppeak = cm h ~ the v c were ml (mb- l) and m{ (mb- l), respectively (p< . ). in a group of subjects ( m/ f, _+ years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb- l and mb- l fell on the identity line. it is concluded that the new mb- l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age ( - ) yrs . they all were connected to servo ventilators siemens c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo , swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with cm h cpap for at least one hour. we turned them to psv with inspiratory assistance (psv cm h ) and after rain we applied psv cm h , and after min psv cm h . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< . ) . respiratory rate (rr) decreased significantly (p< . ) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do ) decreased ns, oxygen consumption (vo ) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo and pvo values in a group of patients, following open heart surgery. methods: upon transfer to icu, patients ( males and females) of mean age _-+ years, were randomly assigned to receive (n= ), (n= ), or cm of peep (n= ). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of ml/kg. the fraction of inspired oxygen (fio ) was adjusted to keep a pao around mmhg. mixed venous po and svo were measured at min, and hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at hours post-extubation. differences at each study time were analysed by anova. results: mean svo and pvo values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo and pvo ) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from to ml, at rr from to bpm, while closely monitoring cpap airway pressure swings. at v v of and ml the rr was limited to bpm. for comparison we explored aps of a one l balloon cpap system, the cpap mode of the puritan bennett , and siemens ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one l balloon system was linear over a range from . to . l, with a cpl. of . l/em h .the cpl. of the l balloon ( . l/em h ) was linear between a volume of and . l. apf of the weighted balloon system was under em h at all v r (except at a v r of ml aps was . em h ), while the apf in the l balloon was up to em h . apf witli human volunteers with the two commercially available ventilators in the cpap mode was about cm h ; while under identical conditions apf in the l balloon system was . emhzo; and in the two l balloon system was below lcm h . conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co (etco ) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco -etco variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) ( + ml vs + ml, p< ) two individual behaviours were observed on tl in pl: four patients presented low etco: (range - mmhg)and normal pacoz (range - mmhg), while three patients had normal etco (range - mmhg) with high pac (range - mmhg). one hour before stopping ilv (t ), vt, etc and paco were the same in each lung. the pao /fio: ratio improved in all patients from the beginning ofllv cst of pl was + % of the normal lungs' cst on ti and improved to . + % ofnl's cst on t (p< . vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco and of the differential paco -etco gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med :$ , ) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from to % of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav %), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an month period. we compared assist/controlled mandatory ventilation (s-cmv, patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, patients) and biphasic positive airway pressure ventilation (bipap, patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation ( . h, p< . ) than patients treated with s-imv/-psv ( . h) and s-cmv ( . hi. with s-cmv . % of the patients required single or multiple doses of midazolam but only . % in the s-imv-/psv group and . % in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group ( . mg) than in the s-imv/psv group ( . mg, p< . ) and in the bipap group ( . mg, p< . ). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p< . ). after extubation the paco patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~ +pst, rs (bates) [ ] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h -h log t (hildebrandt) [ ] where h~ and h are parameters depending on lung volume, and initial time is s for analytical reasons. materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. was , -+ . , and using eq. was . + . . the values ofz~ (eq. ), however, decreased with increasing the tidal volume (vt) according to the following equation: "~ = . - . v t, similary, the values of h~ and h increased with increasing v t according to the following functions: h~ = . + v i and h = . + . v t. conclusions: the behaviour of "% of eq. suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range ( - ml/kg) in ventilated patients. infect this model predicts that "c is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r obtained by fitting an exponential for data of eq. , is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r wh t lsms ira tlmeand r given by:pv~c.~ = d~( -'e-~ )[ ] ere " ' p" tory " and "r are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c and r which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z + e~t.r= [ ] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~ by analyzing the time-course of ptr according to eq and next determining r according to eq. , using the expedmental values of p,i=~, ~ and ti, as well as "~ obtained with eq. . materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. was . . with v t of ml/kg, the mean values (+ sd) of ': and r of the subjects amounted to . • . s and . • . cmh i "~ s. with the traditional multi breath method the corresponding values were . + . s and . _+ . cmh i " s, respectively. with the t-test the difference between new and traditional "~ was statistically significant, between new and traditional r was not significant. conclusions: with the single breath method it is possible to compute ': and r . the mean values of r with v t of nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during - . a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. ) post tb respiratow failure ( %) ) neuromuscular diseases, ( %) } undiagnosed sas { %) ) cope) ( %) ( patients had a overlap syndrom). the programme consists of : ) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). ) group and individual respiratory therapy ) mobilization ) nutritional support ) educational classes for the members of the family. three from the patients passed away (during the year), are under nippv during night with or without supply, pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao /pao ) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients ( + yrs) suffering from ards (lung injury score > . ) for hours or less entered into the study. irv (volume controlled, decelerating flow, % inspiratory pause, lie = / ) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= / ). these two modes were applied for hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume ( . • . ml/kg), respiratory rate ( • "bpm) mad fit ( • %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after , , and hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a -year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * ( . rr of patients examined showed moderate to severe desynchronization. in of these patients a (re)activation of the hbr was found. the strength of hbr amounted to + %. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined patients after open-heart surgery and patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in of intubated patients, of which showed cs. in of these patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining patients. in of these patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k .w. the constant tube coefficients k~ and k are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k are changed rendering the p~,ch calculations inaccurate. therefore, k and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to %. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [ ] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [ ] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be inter- gil cano a, san pedro jm ~, sandar d, herntndez . , carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: ) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and ) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than mmhg or an absolute decrease in systolic blood pressure below to mhg within hour of intabatian. patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h ( ), cardiac arrest ( ) . there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < . ). high paco before lrasheal intubatian ( . - mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < . ) in patients who developed h. paw. ) thexe was a good relatienship between h and high arterial paco before traqueal intahatian and its fast "washing" with mechanical ventilation. ) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit ( ). application of ips has been shown to partly compensate this added work ( ). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd ( ). therefore, atc has been developed as a new form of assisted spontaneous breathing ( ), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls , dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls was connected to an artificial trachea (at, cm long, mm id). the at was intubated with three different tubes of . , . , . mm id and connected to an evita ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of and cm i and atc all with a peep of cm h . the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/ are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<. , **-p< , ns = non significant, f versus s neveltheless, in / patients, invasive ventilation was necessary in mean . _+ hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps & , clinical signs (rr, spo , heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : .a low value of pac on admission until the patients were intubated. . an increased level of cpk in relation with an acute myocardial infarction ( / cases in the failure group, vs / cases in the success group, x~(with continuity correction) : p<. ). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco (< mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators ( , ). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) ( , ). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls , driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls was connected to an artificial trachea (at), intubated with an . nun et]', end connected to one of four ventilators (servo c and servo , siemens,-elema, sweden; evita , driiges, liibeck, frg; pb ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips and mbar; trigger set at maximal sensitivity, peep always mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied paralyzed cppv ventilated ali patients (lung injury score = . • that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve c), first during cppv and then within the th psv hour. airway pressure and flow signals were recorded (cpi bicore) for subsequent analysis. an airway pressure plateau was defined as a flow tracing in which airway pressure was stable for at least . sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; ) during psv inspiratory plateaus are longer than the expiratory ones; ) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = patients). we selected ali patients (lis= . • in vcv mode who had available a chest ct scan performed within days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao > mmhg, irrespective of fie (peep < cmh ). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate < bpm, an increase in fie lower than . compared to vcv, a pace increase < % of vcv value and hemodynamic stability were maintained during the next hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with ventilated patients, after head injury and with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep ( - - cm h ). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: . + . for peep , . + . for peep and . + . l/rain for peep . simultaneously the bioimpedance device recorded a significant increase in cardiac output from . + . for peep to . + . l/mi for peep . (p < , ). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of patients who had been hypercapnic (paco > mmhg) for at least days (range to days) before the end of cv. the psv trial was started as soon as pao was > mmhg, irrespective of fie and with peep < cmh and the psv level had to be < (pplateau-peep) as measured during cv. pace , pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: ) . ) weaning is more difficult in pts with head injury(p<o, ) and iss> (p<o,ol), in elderly(p<o, ) and in pts who need fi > , (p<o, ) and peep>io cm h (p<o, ). ) pts with iss> need longer duration of mv (p<o,ol). ) the mortality rate is higher in pts with iss> (p<o,o ), with head injury (p<o,ol) and in elderly (p<o, ). ) paradoxally, compliance was found to be higher in pts> years than in pts< years (p<o, ). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in patients (individual analysis for each of the eight lungs) at peepe levels: peepe = cmh (zeep) and peepe amounting to %, % and % of the peep• at zeep ( . • cmh ). tidal volume was kept constant ( • . ml/kg). each lung section was divided into zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: pts with acute (a) (paco = • mmhg, pao /pao = . • and pts with chronic (c)(paco = • mmhg, pao /pao = . : . ) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh *s/min), which is an index of oxygen muscle consumption. measurements were collected, after rain of stabilization, at and cmh ps and at two different peep levels ( and cndd conclusions: during psv: ) breathing pattern and ptp are different between a and c; ) in both groups peep does not modify breathing pattern; ) peep reduces ptp in c but not in a; ) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p . and rr. changes in p . were remarkable, homogeneous and highly significant (p<. ), while changes in rr were less pronounced, inhomogeneous and less significant (p<. ). it seems therefore that p . is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. sets of measurements were obtained in each of ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to % of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=. .ctotlsf+l. , r =. ), and with cqs (cqs =. .ctotlsf + . , r =. ). the average difference between ctotlsf and cqs, yet, was much lower (+. + . ml/cmh ) than the one between ctotlsf and cdyn (+ . + . ml/cmh ). rtotlsf correlated much better with rmax (rmax=. *rtotlso+. , r =. ) than with rmin (rmin =. .rtotlsf +. . , r =. ). the average difference between rtotlsf and rmin was + . + . cmh / /s, while the one between rtotlsf and rmax was +. + . , confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. sets of measurements were obtained in mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all reference measurements, rcdyn (ve/v'epeak= . orcdyn-. , r =. ), rcstat (ve/v'epeak = . rcstat -. , r = . ), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period - we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao /fio < , despite maximal ventilator settings: peep > cm hz , fit > . . a total of patients matched these criteria, males and females with a median age of ( - ) years. seventeen suffered from severe trauma. chfjv was started following a median period of ( - ) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit . , pao /fio , peep cm h peak airway pressure (pap) cm h . chfjv consisted of high frequency jet ventilation with a frequency of to breaths/minute, driving pressure of . to . arm, and inspiration time of to percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l to breaths/minute and tidal volumes of to ml. results: following two days of chfjv of patients showed an improvement of ventilatory parameters; peep could be reduced to < cm h in patients, the pap was decreased with > cm h:o in patients, fio could be reduced to < . in patients and finally the median pao /fio ratio changed from to . during chfjv patients died, of respiratory failure and due to multiple organ failure, died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm ; ( ) :a ). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with groups to ventilate nonperfused, lavaged rat lungs with small vt ( to ml/kg) for hours and studied the effect on compliance and lung injury. group : peep>ping group : peep<pin f with an si, inflation to on h over sec, to recruit volume; group : peep<pinf without si; group : control group, lungs were inflated at peep<pin f, but not ventilated. results: in group and group static compliance did not change after ventilation (table) . in group static compliance fell significantly. group showed significant changes in the pv curve, which were less severe than in group . results from morphological examination are pending but preliminary results showed less lung injury in group , compared to group . conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy ( ) minimizes lung injury as well as, or better than using peep above pint" and ( ) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles eeg examinations with fourier spectral analysis' of patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co and blood gases during weaning. results: patients had invariable eeg activity or short -waves period (till one hour). the weaning of this patients was fast arid sucsessful. other patients have had a decreasing of a-activity, an appearence of -waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u , hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett ) but not others (siemens serve ). we studied the two types of systems in two groups of patients mechanically assisted with pressure support ventilation ( + cmh ). in the first group (pb ) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt ( + cmh .s/min - vs + , p< . ). by contrast no significant difference appeared in the second group (serve ), as predicted by the bench study despite marked interindividual differences ( + cmh .s/min - vs + , p = . ). we conclude that ) rigorously performed bench studies can predict in vivo effects, ) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs , lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: ( arf and ards) sedated paralysed patients were studied using a serve c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to - ml or a maximum paw of cm h (alfi). the quasistatic elastic pressure (pel,qs was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v from up to - ml were followed by a s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~ i~: in ards, mean cst was . + . and cqst . + . ml/cm h (us), lipst . + . and lipqst . + . cm h (us), uipst . + . and uipqst . + ~ cm h (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, ! ). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > colony forming units / ml. results: for the patients studied, the mean saps score (+ sd) was +_ , the initial pao /fio ratio was -&-_ , the duration of mechanical ventilation (mv) was + days. the mean delay before the onset of the first np was . + . days ( - ), and the mean pao /fio ratio was +- . respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in % of the patients studied. alteration of gas exchange was present in of the patients ( np) . a new pulmonary infiltrate was present in only np ( %). an increase of fever was noted in patients, an increase of leukocytosis > % in patients, an increase of volume and purulence of sputum in of the patients with np. the degree ofgazometric worsening (pao /fio before np minus pao /fio during np) during the first episode of np was + mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was in / patients, ( / ), ( / ) or ( / ). two patients only had a pulmonary colonization (ptc: < cfu / ml) before the first episode of np. the incidence of np is high ( %) during severe ards. the first episode occurs in average:at the th day, and is the cause of a severe hypoxemia (pao /fio ) . the onset of a np may contribute to the high mortality rate observed in our patients ( %). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of . • j/l, a mean value of inefflency, ~ of . ~= . and a mean value of resistance, ~, of . • cm h s/ . the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ---- . • . cm h s/l. during methacholine provocation~ ae rises more than five-fold up to . • j/l~ doubles to . • and t~ increases to a maximum of • cm h s/l, with armax : . • . cm h s/ . the variation in rk becomes more pronounced with higher doses of methacholine. methods: ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo , and aado decreased while paco increased and tda vo ume and co remained unchanged. on the contrary, in group v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo and aado increased with constant co, the pressure support level needed to achieve the target rate was much lower in group than in group ( , -+ . vs . _+ . ). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, - years)breathed spontaneously through an uncut ett of . mm id via a mouthpiece. the ett was connected with a prototype ventilator evita modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, ( ) asb ( mbarover mbar peep), ( ) atcin, ( ) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+ ), ,,equal" ( ) or ,,worse" (- ). inspiration and expiration were investigated separately by presenting mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within hours after cardiac arrest and repeated , , , , and hours later. results: we studied patients, females, mean age , + , years. the pi decreased s!gnificantly between th~ first and the last measurement from . _+ . to . + . (p = . ). cerebral oxygen extraction ratio decreased also from . + . to . + . (.p = . ). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels hours later, rasp. . pmol/ml (median; th . - th . ) and . pmol/ml (median; th . - th . ) (p = . ). eighteen and hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = . , p = . and r = . , p = . ). we.also found hours after the first measurement a significant correlation between pi and cgmp levels ( r = . , p = . ). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology ( , ). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged - years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m segment of the middle cerebral artery (mca). results: a total of phases of vf were investigated. duration of vf ranged from to seconds, with cbfvmc a (mean_+sd, cm sec - ) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: icu nurses were asked to inflate a testlung model times with a specific air volume ( ml, ,ml or ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated ml ( ml). the mean inflated volume for this group was ml. when the icu nurses had to inflate ml the mean absolute volume difference was ml with a mean inflated volume of ml. inflating ml produced an absolute volume difference of ml with an mean inflated volume of ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the ml and ml for all specific air volumes. % of the last inflations with feedback were significantly smaller than ml from the specific air volume (p < . ). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when ml and the smallest when inflating ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in domestic pigs. following rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for min. electrical defibrillation was then attempted and of animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of . ml/kg to . ml/kg (p< . ), and left ventricular stroke work index (lvswi) from . to . mmhg,ml/kg (p< . ). both svi and lvswi remained depressed for another hours. these decreases were associated with increases in heart rate from bpm to bpm (p< . ). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d- bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach - % depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from - min, mean min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after - min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report cases of prehospital cpr with primary asystolia terminated after - rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after - min and cpr efforts were immediately resumed, nevertheless, of the patients died at the scene, while could be hospitalized with stable circulation. one of them died hours after admission to the icu, the other survived for weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [ ]. we reported our results on such monitoring in [f.coaobbeb,r.fe enb~-kap~monorm~, ,n ,p. - ] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po ,pco (mm rg),so ,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po +pco ) in mm hg in ap blood. these data were derived from:i) subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [ j. in control group it is equal to , • mm hg. tests on sgp neither exclude nor substitute conventional (pc and pco ) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: ) using towel clips to reapproximate skin only, )i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, ) using loosely tied retention sutures for partial closure, ) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from to . all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died t days later of multiple organ failure. there were no complications related to the closure technique in any of the patients. conclusions; . selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from / through / , pts underwent open heart surgery at our hospital. of th~se, pts ( %) (age _+ yrs) had a cardiac arrest during the first hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) ( pts), valve replacement (vr) ( pts), cabg and vr ( pts) and aortic aneurysm ( pt). the preoperative ejection fraction was _+ % whereas bypass and aortic cross-clamp time were + and + rain, respectively. prior to arrest, they had a cardiac index of . _+ . l/min/m and were receiving . + inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation ( pts) and bradyarrythmia ( pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in pts. eighteen pts ( %) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t pts, %), tamponade ( pts, %), rupture of the proximal vein gra& anastomosis ( pt, %), graft occlusion ( pts, %); no cause was found in pts ( %). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (- % incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of doctors and nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between - . after the first screening we held a x hours training. doctors and nurses were trained for the endotracheal intubation (group it , t ) , doctors and nurses were trained to use the laryngeal mask (group lm , lm ) . all respondent were trained to use the bag-valve-mask device. day, month and month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. x hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and % responded the course "very useful". . % of doctors and . % of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies ( . %), penetrating abdominal wounds with injuries of mesenterial vessels ( . %), injuries of the liver ( . %), blunt abdominal trauma with lien ruption ( . %). in . % patients had the previous somatic pathology. blood loss volume was - ml, & the reihfuside blood volume was - ml, consisting - % of blood loss. it was needn't to fransuse donor blood in . % in further but - ml of contanined erythrocytes were frasfused for supporting of hb concentration on the g/l ( g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ functional indices of cardiorespiratory function were evaluated. results: pregnant women with ~h-gestosis were examined before and after i/v infusion of i ml of . % clophelin solution, . mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by %, of the heart rate by io. %, of the oardial output index by . %. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for patients. the mean iss value was . + . while the median value was . minor injuries were present in ( %) patients with iss less than , while ( %) patients with iss more than had severe injuries. increased mortality of the older patients was noted in the range - . all patients older than died while % of patients below yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp- ). results : fifteen patients were admitted in icu and in a medical unit. fifteen patients died. a group of patients had a lactatemia up to mmol.l" . in this group of patients, had acidocetosis, had asthma, had cerebral vascular ischemia, had neoplasia, had cardiogenic shock, was epileptic, had congestive heart failure, had acute respiratory failure, had septicaemia, had hyperosmolar status finally had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( . • vs. . + . , p<o.oool, respectively), the cut point of lactatemia was . mmoll" se was %, sp was % and the accuracy was %. on the other hand lactatemia was higher in patients admitted in icu than in medical unit ( . + . vs. . • p<o. , respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over months. setting: a -bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < x , neulropaenia < x , and lymphopaenia < . x ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days , , and . the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < . ) and hollow visceral intra-abdominal injury (p < . ). eight ( %) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < . ) and significant differences were found in absolute neutrophil counts (p < . ) and differential percentages (p < . ) on days and . no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that . % traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: . besides a physician specialized (vii --+ vii -~ viii gradus) in urgent medicine, it's necessary to introduce two ( ) medical technicians instead of one as is existing now ( ~ ). .the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). .the technicians have been specially educated for urgent cases (iv ~ v ~ vi). . such a new team structure should be more capable in professional sense. .we can see an importance of team-work in ects. . such teams should also be more economic in comparison with existing one. . we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis ( dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc , dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at - mg of cells per i kg of patients weight. dialysis was performed during - hrs. patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: . ml of hemosorbent and . ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of - ml/min, during - min. average volume of hemoperfusion made . . conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam ( . mg/kg),thiopentone ( mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone ( mg/ kg/die),mannitol ( .smg/kg) and diphenylydantoin ( mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in patients ( m., f., + y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is , - , mmol/l. although more than % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in % of patients ( ) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from st of january till st of december . results; in this four years period there were abdominal injures. there were blunt injures. among them in patients peritoneal lavage was applied. results were positive in patients and negative in patients. false positive finding were in patients and false negative in two patients. in patients with positive lavage there were more than , /mm red blood cells, more than , /mm white blood cells and the level of amylase was up to u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than , /mm red blood cells, less than /mm white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se en md, z.milo~evi md, *s.srdi md, k.sar ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the st of january till st of december , are presented. results: among operated patients there were females ( . %) and males ( . %). there were patients ( . %) with gastric bleeding and patients ( . %) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in patients ( . %), ulcer suture in patients ( . %), billroth i in patients ( . %) and billroth ii in patients ( . %). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in patients ( %), duodenotomy with ulcer suture was done in three patients ( . %) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj b ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in ( %) patients, while in ( %) it appeared unaffected. cpk-mb of % and more in comparison to total cpkwas . + , % in patients, and , + , % in pts (p< . ).c of . + . l/min in patients with cardiac contusion, measured hrs after admission at the latest, was significantly lower than in group of patients with co of . + . l/min (p< . ). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was % lower than in the blood inflowing. hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was % lower compared to the vb and did not change for hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent years patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. ( . %) underwent urgent surgery, of them ( . %) with cardiogenic shock and ( . %) with massive pulmonary embolism. patients ( . %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in cases ( . %), in ( . %) patients subsequent pulmonary embolectomy was performed. mortality rate was ( . %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and ( %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in % patients. pe mortality rate is repoted to be %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr ),free thyroxine(ft ),total tri-iodothyronine(tr ),free tri-iodothyronine (ff ) levels were measured within hr.of intensive care unit admission(first day) in multiple tratmmtized male patients(injury severity score higher than ). in fifth day the same hormone levels were repeated. tsh,et ,ft ,tr and ft levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt ,fr ,et and ft in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm ( ), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( , + , mmol& norm - , + , mmol/i ), whereas rats without glucagon had urea content , + , mmol/ ( p < , ). osmolarity of blood plasma in rats having glucagon injections was , + , mosm/kg h , without the drug - , + , mosm/kg h ( p < , ), ldh activity - , + . and , + , ( p < , ) mcmol cec- accordingly. lethality in the group of rats without glucagon was % /n= /, with glucagon - (n = ). in patients with hypovolemic shock in minutes after glucagon administration stroke volume increased to % (sv), miocardium contractility -to % (mc), peripheral resistance of vessels ( vr ) decreased to %. in - , hrs sv increased to %, mc-to %, pvr -decreased to %. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing, ), p. n, vasina (t) ,n.sebbota hph ( ). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k r-key, [ maine ( ), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i ~ mmol/l down to # mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal z, there of i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in % and morphologically in - % of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last years patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were patients with acute myocardial infarction treated before hospital by the cardiological groups. patients were treated with the early infusion of nitroglycerin and without. the results of treatment were: in the group with infusion: mortality in the first hours % the full disappear of pain % in the group without infusions mortality in the first hours % the full disappear of pain % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used mg prazosin tablets sub lingua and measured blood pressure ones during hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was . the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in % of patients. the general condition of patients became better in - min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: patients, aged _+ years (mean _+ sd), and burnt _+ % of body surface, were studied from day (d ) to d . serum samples were collected at do, serum and urine from d to d , and on dio, d , d . cutaneous exudates were extracted from the textiles surrounding the patients, collected over h periods from d to d , using bidistilled acidified water. mg supplements ( . to mmol/ h) were prescribed from d to dio according to the severity of hypomg (serum mg < . mmol/i). results: mg serum levels decreased below reference ranges in patients between d and d , and normalised thereafter during supplementation. urinary mg excretion was increased in patients, mean excretion being . _+ . mmol/ h until d . mean daily mg cutaneous losses, were mmol/ h, i.e. mmo; in days. large inter-& intra-patient variability: the daily exudative losses ranged from to mmol/ h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t / ,id ). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for seconds with ~ hot water to achieve a % tbs partial-thickness burn. blood samples were taken prior and after surgery as well as , minutes, , , and every further hours after thermal trauma. two control groups (each n= ) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n= ) received c -inhibitor concentrate at an initial dose of u/kg body weight either immediately or hours after thermal trauma (n- ), followed by three further applications (of reduced amounts) every hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin- (il- ) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of multiply injured ( ( ) ( ) ( ) il- (nis) ( ) ( ) ( ) il- (nin) ( ) ( ) ( ) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il- is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no ) and nitrates (no ). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included patients admitted to the intensive care unit of the burn center of brussels (age : - years; burned surfaee area - %), and control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age - years). the patients in both groups were fed enterally with to kcal/kg/day of a standard solution. in the burn group, daring the study period, patients were mechanically ventilated, required vasopressor (dopamine) therapy, and received antibiotics; patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no /no determinations (griess reaction) daily from day to day (n=ll) or to discharge (day , n= ; day , n= ) in the burn group, and once the control group. in the burn group, no /no levels were elevated at day ( _+ #moles/i), reached a maximal value on day ( - p.moles/l) and progressively decreased to day (fig). these values were higher than in the control group ( . :t: . #moles/l, all differences p< . ). however, no correlation was found between the plasma no /no levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no /no levels than those who did not ( + vs - #moles/i, ns). the present study indicates that burn noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no /no plasma levels, which are ao so consistent with an enhanced no pro- o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics ( ). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury ( ). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa , male, female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a gauge needle was inserted into the lumbar subarachnoid space at level l /l and connected to a trancducer (mx | medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco constant. after a resting period of minutes basline measurements were performed including csfp, cvp, map, hr, etco (cardiocap | datex). then the volunteers were given urapidil . mg/kg over a period of minute. minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p< . significant; values: mean_+sd, si = mmhg. - _ " _+ * _+ * - _+ " _+ csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map ( ). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of -min ischemia, reaching peak at . h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied patients with severe head injury (glasgow coma score < ) admitted to our icu between fobmaly and december . icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of patients were additionally icpc monitored by a fiber optic transducer (comma r~), and seven of patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = . (p < . ) with a regres~on line y = . + . x. four of ~hx lcpc -icpv patients had r > . when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = . (p < . ) with a regression line y = . + . x. corralalmu eoetfieiont was inwer than . in all seven patients. corrdation eoelfieients for levals of icpv > man hg, > mm hg and > tuna hg with icpe showed r = . , r = . and r = . respectively; and with icpe r = . , r = . and r = . . the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors ( , ). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam ( - mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam ( - mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, patients aged over years suffering from head injury, with glasgow coma scale (gcs) of or less, were studied retrospectively. twenty-six patients ( . %) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three ( . %) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs ( . vs. . ) and median age ( . vs. ). the incidence of brain pathology in the two groups was the following: epidural haematoma . % vs. . %, acute subdural! haematoma . % vs. . %, intracerebral haematoma . % vs. . %, subarachnoid haemorrhage . % vs. . %, diffuse haemorrhage . % vs. . %, contusion . % vs. . % and non-visible pathology (normal ct) . % vs. . %. unilateral pupilary dilatation was found to be . % in group a and , % in group b. the mortality during hospitalisation in the i.c.u. was almost the same: % iu group a and . % in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a month period ( - r the icp was monitored in p~,tients ( male, female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was . years ( - ). the glasgow coma scale at admission was . ( - ). in all cases the device was placed wfihln the first hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was . days ( - ) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c ntra!s by ct-scan. in case an intracranlai hemorrhage was detected and drained. the overall survival rate was . %. showed a complete resolutl n, in other . % psychological residuals like decreased mentatlon, in . % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and . % of the injured remained in a comatous status. in % of our cases the measured values correlated with clinical course and management. in cases ( . %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality . identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age . _+ . , injury severity score . + . ) admitted to icu were studied. incidence of arf was . % ( / ). arf was defined as persisteat plasma creatinine > mg/dl with or without oligoanuria . arf was defined as early when occurring within the first hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in patients while larf developed in patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis ( . % for both) were observed in larf group, when compared to earf ( % and % respectively). abdominal trauma was more frequent in earf group ( % vs %). the gs for earf and larf were respectively _+ . and _+ . while in the group who not developed arf (narf) the gs was . • conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher ( . versus . ) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc ( ) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied consequitive patients ( in group i) and ( in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was min. in group i we recorded r.o.s.c. (return of spontaneous circulation) , %, death %, continuation of cpr efforts , %. while in group ii, %, %, and , % respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: patients, aged _+ yrs (mean_+sd), with burns covering + % of body surface were studied from day (d ) to d post-injury, were randomised in groups (g): g -control receiving recommended te supplies + placebo; g -receiving in addition large supplements of cu, se and zn from d to d . enteral nutrition was started within hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d and d . infectious episodes and micro-organisms were monitored until d . results: the patients' characteristics were similar g & g . the total leukocyte counts were higher in g between d and d , due to increased neutrophils (significant from d to d ). total cd + and cdlg+ cells did not differ, whereas cd + (monocytes) were significantly increased at d . proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g with a mean of . _+ . infections during the first days versus . _+ . in the control group (p < . ). conclusions: the large te supplements for days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of % body surface area iiib-iv burn in rats. a burn eschar was excised on the rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the th day after burn infliction the activity of cathepsin d in injm'ed muscles is times lower under an adsorptive dressing than under a gauze bandage (control) (p< , ), the activity of trypsin-like proteases is . - . times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is - % and - %, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first hours icu data and icu admission had to occur within h of traumatic insult. over the last years, pts with severe head injury (gcs< ) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of . h after trauma, range between - h). in a total of pts (= . %), jugular bulb desaturatiens (< %) were noticed during this early h period. in pts (= %), jugular bulb saturations higher than % were observed, whereas pts (= . %) revealed no abnormal sjo data ( - %) during these first h. concerning the periods with too low jugular bulb saturations (n: ), we found the following correlation ; in pts (= . %) cerebral perfusion pressure (cpp) was below mmng, in pts (= . %) paco~ was below mmhg and finally in pts (= %) we found primary intracranial hypertension. for the high jugular saturations (n: ) we found a primary intracraniaf hypertension in f pts (= %), and a pace level above mmhg in pts (= %). in all patients we could restore jugular bulb saturation within normal range ( - %) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury ( ). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp> mmhg) and cerebral hyperaemia (sjo~> %) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of head injured pts received treatment for intracranial hypertension over the last months. six of them met the criteria set for the administration of indomethacine. in pts, no decrease in icp or in sjo was observed and both pts died due to therapy refractory intracranial hypertension. in the other pts, a significant fall in icp and in sjo was observed shortly after indomethacine administration. in pts we observed a catastrophic fall of sjo= even below %, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the pts, icp remained under control without further administration of indomethadne, but he died days later in multiple organ failure. the other pts, needed multiple indomethacine administrations (for pt even during consecutive days) to finally control icp. in all pts, icp was finally controlled, but only pt survived. both other pts died from systemic causes (multiple organ failure in pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in consecutive pts, suffering from severe head injury (gcs< ). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo information. pts revealing ich and sjo=values above %, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than %, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=> %). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed pts with ich, with an overall mortality of . % due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer pts, csf drainage alone was sufficient to control ice of the remaining pts, pts received mannitol and pts were hyperventilated as second approach. in the third line, pts were managed with barbiturates, with mannitol and pts with hyperventilation. finally, barbiturates were used as the final rescue in pts. these results reveal a less frequent use of mannitol as only pts received mannitol, compared to the pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier ( pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo -guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po , icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in piglets ( days old, , - kg) of either sex we produced a moderate brain injury ( , arm., msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and min. after brain trauma and after , and hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to mm hg being highly significant (before mean i mm hg, range - mm hg) could be observed: mean arterial blood pressure rose from appr. mm hg to ii mm hg for - min. in two animals this was followed by an ischemic period lasting min. accordingly icp values gradually returned to starting measures within hours; in the ischemic animals they remained at a level of about mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within hours. cerebral blood flow (ldf) fell from mean values being i before trauma to appr. zero and recovered to around . brain po started at mean values of mm hg (range - mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: ) suction of gastric contents from trachea during intubation, ) presense of a predisposing factor, e.g. coma. ) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale , because of cerebral injury, and patients who died within days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were and the non trauma patients . from the trauma patients, had cerebral injury and were polytreumatized without cerebral damage. from the non trauma patients, had malignant neoplasms, neurological diseases in terminal stage, old age, drug overdose, and several diseases. eighty seven from trauma patients ( %) and from non trauma patients ( %) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the groups (p> , ). the severity of complications was also proportional in the groups. eighteen deaths were recorded in the trauma patients (mortality %). only deaths correlated directly or indirectly with the aspiration ( %). in non trauma patients, deaths were recorded ( %). twelve deaths were recorded in patients with neoplasms, deaths in patients with neurological diseases, deaths in aged patients, death in drug overdose patients, and death in patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< , ). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p< , ). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in groups. the patients of group a ( patients) received fluconazole rag/day for days. and the patients of group ( patients) amfotericin-b rag/day for also days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from of the group a ( %), and from of the group b ( %), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p< , ). all the patients before treatment had positive for fungi blood cultures. after days of treatment, patients of group a and none of group b had positive cultures. eight patients (from who had positive cultures of bronchial secretions before treatment) of group a. and (from ) of group . had positive cuttures of bronchial secretions after days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p< , and p> , ): ten patients (from ) of group a and patients (from ) of group b had positive urine cultures, after days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p< , ). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in patients, chills and fever during infusion in patients, and hypokalemia in patients). three patients of group a and patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were . all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, in group b, in group c. and in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the groups as to number of complications ( twenty four patients had chest complications in group a, in group b, in group c, and in group d. p> , }. the duration of mechanical ventilation was not statistically different among the groups (the mean duration was , days in group a, , in group b, , in group c, and , in group d, p> , ). there was also no statistical difference in mortality among the groups (six patients died in group a. in group b, in group c, and in group d, p> , ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in trauma patients admitting between december , and july , in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in ( %) of survivors and in ( %) of non survivors .we noted low fncidence of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.( . vs . , . vs . meq/l, p< . )the duration of hyperlactemia also was higher in the patients with organ complications. ( . vs . days, p< . ). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.( . vs . , . vs . meq/l, p< . ) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj , kjdo and ceo in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and jo monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous hours, normal x ray chest and within the first hours from injury, were considered candidates. peep value where increased fro~ zero to cmh , in three different steps, each one with min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo , in the case of hemodynamic deterioration, during the study, ppc ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. patients were candidates, of them were exc;uded due to haemodynamic instability, k total of completed all steps and were e~amined, men and women, age ! . yrs. at hospite; arrival, glasgow were < in patients, and > in the rest . patients mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied patients( females), aged (mean-..+-sd) . _ . yrs, which were consecutively (from august to march ) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was . -+ . (range - ). the following data were evaluated: ) number of medical departments, where health care was provided until final admission to the icu, ) ambulance transportation conditions, ) catheters and tubes inserted before admission, ) vital signs upon admission ) information provided by referring physician (scored on a to scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), ) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu ( : . %, : . %, : . %, : %). thirty/ ( . %) patients were escorted by a physician. twenty-six/ ( . %) were transported on oxyge n, fio (mean__.sd): -+ %, pao : . -+ . mmhg. five of the remaining , for whom no oxygen was provided, had pao : . -+ mmhg. twelve/ ( . %) were intubated and ventilated during transportation. thirtyfour/ had a peripheral venous line, / had an arterial line, / had a nasogastdc tube, / had a urinary catheter. eleven/ were sedated and / were paralysed. three/ were on inotropes. vital signs upon admission were: arterial blood pressure, systolic . -+ mmhg, diastolic -+ mmhg, heart rate -+ bpm, temperature . -+ cc. patient information score was --. . . the actual state upon admission was found substantially different, as compared to the description of the referring physician, in / ( . %) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since . % of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from to years. dosing ranged from mglkg to mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from st june to july , all the calls for emergency care were analysed using a questionnaire of items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively % and %. the performance of the model is %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may to april , patients were admitted to our icu with bci ( m, f, age + ). a history of direct trauma was present in patients. admission gcs was in all patients, and was associated with hemiparesis in of them; the last became paretic hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan ( - days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table x in z test. res.cl~s: of patients were submitted to thrombolysts and died. the higher incidence of bracb, ar~lhmias (ii degree gg p t e and av block. i degree av block. avsb . <ii. sinus arrest) that requited the insertion of avsc . <cl temporar~ pace-maker was recorded in group a b vs c . ns in % of the cas ~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and , %nacl/ %dextran . methods:in polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q ) prevalid.replacement of the volume loss egan in institutions of general medicine,frequently by rl and dextran or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution dministered ~o the injured wer~ - ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the , %nacl/ %dextran @(groupii)( -sml/kg observed to the final surgical management,had statistically higher map(increase of % in relation to admission), cvp(average increase of , cmh~o),diuresis(averagely omi after min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q %,average increase c~ of cvp , cmh and average diuresis oml after omin). total quantity of the administered solutions was significantly smaller in the groupii( ml: ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl: ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of , %nacl/ %dextran lasted minutes. w.scherzer(l~,k.lechner( ),r.simon( ). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin !( )neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson, ) ,in general anaesthesia (bennet, ) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger, ) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter( ) ,basal stimulation (bienstein, ) , bobath-therapy( ) and facilitation of a physiological moving pattern(pnf)according to knott( ) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: patients (mean age: ) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n= ), acute congestive heart failure (n= ) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of rrd ice-cold glucose % non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of months. the tt is a eompetitve immunoassay which gives qualitative results within i minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study patients were enrolled, in of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of . and specificity of . . conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure > rorohg and diastolic blood pressure > nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below mmi-ig and diastolic blood pressure below mmi-ig within minutes after start of treatment. results: patients were included in our study, ( %) patients responded successfully to treatment. mean arterial pressure decreased in responders by . mmhg and in non-respenders by . mmhg (p< . ). responders and non-respenders differed signii'icantly concerning pra (p= . ), ace (p= . ) and ang ii (p= . ). . . the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r= . ; ang ii: r= . ). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july to june , files of patients consulting to the ed for smp have been retrospectively analyzed. results: patients, women and men, . + years old (range - ) have been admitted for episodes of smp ( % of all consultations) whose relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively . %, . % and , % of patients. the interval of time between the ingestion and emergency consultation was noted for % of smp ( + min, ranges - ). the involved products name was known in totality in % of cases with an average number by episode of . + drugs (ranges - ). the most often, ( %) or ( %) different products were interfered. the nonbarbiturate psychotropic drugs accounted for . % of the products (benzodiazepines %, antidepressants . %, neuroleptics %, carbamates . %, imidazopyridines . %, cyclqpyrrol nes . %). analgesics and nonsteroidal antiinflammatories represented . % of all drugs, anticonvulsants . %, cardiovascular drugs %, antiinfective agents . %, drugs against cough . %, muscle relaxants . % and antihistamines h . %. the benzodiaz pines were present in episodes, alone in episodes. in . % of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in . % of cases, activated charcoal in . % of cases, flumazenil in . % of cases, naloxone and acetylcysteine in . % of cases; orotracheal intubation was performed in patients. admission in hospital was effective for patients, in medical ward (n = ), psychiatry (n = ) or icu (n = ); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately . m is based on the principle of three identical bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate ( ). method: a plaque of electrodes, positioned on the left anterior ventricular wall of anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused ( . mg/kg/min iv) during rain; at t , dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least % of its value at to was demanded before the administration of b. results: dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively ( < . ) compared to values control fro)except qrs at t . the b ( + ml/kg; ranges . and . ml/kg) modified no studied dements outside of the ( }rs. to ti t t t t t a , + , , + , , + , , + , , + , , + , , +- ,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from (--full agreement) to (=disagreement). overall, "/. of the active emergency physicians participated in the survey. results: . % of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only % would not hesitate to go into action by dark. . % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only % would do it. . % of the doctors would rather stop flying. the most common reasons for ,uitting were wish of family and fear of an accident. . % conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. crash landings within less than years with deadcases and badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in patients (age - years). the indication for intubation was elective surgery in patients (asa i-ii) and emergency intubation in patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the emergency patients was dyspnea in , cardiac arrest in , trauma in, and unconsciousness due to drug overdose or seizures in patients. intubation was facilitated with medication in patients. results: of the elective patients, ( %) were successfully intubated. six patients ( %) needed two attempts before successful intubation. the duration of intubation exceeded seconds in patients ( %). of the emergency patients, ( %) were successfully intubated. six patients ( %) needed two attempts, and the duration of intubation was more than seconds in patients ( %). in % of all patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in patients ( %). oesophageal intubation occurred in patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < x e /i. in each treatment the same type (av- , fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between and s. the initial enoxaparin dose was rag every :th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels . and . mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin ( . + . h versus . + h, ns). the mean aptt-levcl during heparin treatment was s and during enoxaparin treatment s (ref. - s). the mean daily dose of heparin was nag, that of enoxaparin lg mg. the mean anti-xa activities were . u/mi and . u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load data obtained through direct measurement from the monitors and the lab, the program calculates parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for pts during aperiod of three .y~m , treama pts were . %), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g =- a p> . ; roc = , ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in dogs -~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made . + . ml/kg). the treatment was begun after .u+o. hrs of h. "sl", created on the basis of-sorblt and natrium lactate ( mosm/l) was injected into v. femofalls at the dose of io. ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to . mm hg and - . + . cm h (p .o ), while heart rate (hr)-increased to . + . per min (p<.o ). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by . % (p<.i) and . % (p~. ), while fibrin degradation products (fdp) enlarged by . % (p~ . ). after - min of treatment termination abp and cvp increased to . + . mmhg and . +o. cm h (p<.o ), and ~[r diminished to t . + . per min (p>. ). at the same time the indtces of pic and pf enlarged by . % and . % (p>.i), while fdp diminished by . % (p>.i). one of dogs survived. life duration of the other dogs was . + . hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo through opljcal fiber and the cerebral flux with tcd. methods: since january until june , we collected ht admitted to the icu, and of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ , with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< , sjo is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , ( m, f) , (m. age: + years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was > in all studied patients. tbe group a ( m, ") had no tremor and/or fever on admisskm, while em group b (tin, the above criteria were ix)sitive. bhx~d samplings were taken - hours after accident and - rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, healthy people were examined. _resu!_ts-il-lct, il-ii~, il- and tnf-tt levels were similar to control group levels in both groups a and b. i!,- and g-csf levels were found increased in both groups (p< jxjl), while il- levels were statistically significant comparing to group a. in con_tin_skin, during immediate post raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than ,. , cannot be detected,whereas .- was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep % burn, cornplicated by bloodshed the of blood grows at - times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to %, and % in hours. in hours after the trauma the-process is accompanied b ! , % lower inhibitory activity of blood, where as at the peak of the trauma it was , ~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and -fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at times; [ emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing - , kgr and at age of - months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and . ml (this volume assuring a normal incranial pressure after kaufman ) of autologous blood was injected into a depth of mm via a needle of . mm bore. the wound was closed and the animals were left to recover. nimodipine, of , mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into % formalin solution. tissue sections of ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was --. ~t while that of group b was + ~t (p< , ) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: pts with - % bsa second to third degree flame burns (respiratory tact injury excluded) to yrs of age were randomised to study (n= ) and control (n= ) groups. burn shock was treated with hypertonic saline -bicarbonate solutions ( mmol/l na +) ml/kg/%bsa for the first hours and ml/kg/%bsa for second day. analgesia in control group for the first hours was provided by regular hourly intramuscular administration of mg of morphine sulphate and mg of analgesic -antipyretic analgin with mg of diphenhydramine (dimedrol). from the rd day regular administration of morphine was finished. in the study group ixg of clonidine was added -hourly for hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, - ), sleeping time, spo , hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first days was . - . vs . - . in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only pt showed sign of aws. mean vas score was in . - . range for first postburn days. pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo values in study group were in - % range, among controls - %; vomiting was absent in. cionidine group vs cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs ( -+ kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter- polysulphone membrane) and remained stable for one hour (pao~= . • peco = -+ mmhg, ph= . -+ . , bp= -+ mmhg and pap= -+ mmhg). all was induced two hours after oleic acid infusion ( . ml/kgr) into the pulmonary artery (poo~= . _+ -p< . , paco~- . _+ -p< . , ph= . -+ . -p< . , bp= -+ -p=ns, and pap= _+ -p< . ). fio % for the next minutes did not significantly altered the b ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter ( l/min) while filtrate and gas were removed from the outlet port (bypass flow ml/min) b) through a thin intratracheal catheter positioned cm above the carina ( l/min). the fio given through the ventilator readjusted to %. results replacement fluids/filtrate during the next four hours were not exceed . l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.= . objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied randomised clinical groups of patients each containing patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the -st and on the -th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c and c fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the -st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the -st day, while on the -th and on the -th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, g. sofiiski str., sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a -year old male patient with extremely massive intraabdominal haemorrhage ( m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of ml of autologous blood using an original autotransfusion system developed by us ( pat. no / . . ) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first days after the trauma in patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over . results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the -nd and the -th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity ( + pg/ml compared to + . pg/ml in control) were found od day following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages h following trauma ( - % compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase ( %, h following trauma and % and % on days and after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days and following trauma, and initially but persistently high levels of plasma tnf alpha in da rats ( - fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, , iso , yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca - t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least wounded and sik people. in federal republic yugoslavia, it was proposed such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < % may be more dangerous than short-term levels of - %. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was / and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of % was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef %). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a year old male ingested mg doxepin (sinequanr), a tca and mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs / ), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of b.p.m., an arterial blood pressure of / mmh g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively gg/l (toxic level #g/l) and i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed / h later. nahco ( meq/ h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of patients treated" during a two year period ( - - to - - ) in our i.c.u who meeted the following criteria: ( ) coma score _< gcs within the first hours, ( ) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes - ), ( ) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ meq/l). survival was defined up to the th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = . ). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in [ ] the pre-existing equipment of the volunteers was completed with type a ambulances and special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, consecutive emergency interventions in the time period from novembre , to april , . the emergency physicians treated male ( %) and female ( %) patients, patients died before hospital admission and patients ( %) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period year earlier (november to april ) without attending physician the volunteer rescuers transferred all first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate ml/min in the recirculatory mode. its duration was - min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products - times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e tabli~mczr bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, yejin-l)a-l)ao, wuhan fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving ( . ~), the successful rate of saving tra ma ( .~), the suscessfut rate of saving shock ( .~) and the successful rate of cardioputmonary resuscitation ( . ~) daring the three years after t he network operated with these before ( . ~), ( ]. ~), ( . ~) and ( ft. ~), the successful rates after operating were remrk~iy higher ( p<i).o ). conctusions~ the above results showed estabiishmnt and mena emont of region at medical first-aid network had very si~nificont action during mergentty s avin the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med ; s. nouira. chest ) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among consecutive victims of se presenting to the emergency department, were managed using sav (group sav+). these patients were matched to envenomated patients who did not receive sav (group sav- chest injuries are the cause of death in % of trauma fatalities and a major contributing factor in an additional %. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january to december , all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao /fio < ) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: severe trauma patients were considered. of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais< ). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. patients with severe thoracic trauma (ais>= ) were admitted into the study. the mean iss was . ( - ). thirty-six patients required artificial ventilation for at least hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in cases pneumothorax was diagnosed an the initial cxr more patients had a pnx which was identified only on the ct. in cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, % ( / ) presented a pnx that required the insertion of a thoracic drainage. only one third ( / ) of the pneumothorax could be recognised on the initial cxr, while other were decompressed before performing the cxr. as many as % of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a % pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli , .~osid, m.zesevid, m.risovid, d.stepi , d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod: a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given - prcpofol bolus doses (o, ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion ~ ~ropafol. as sdflitianal were given fan-i~l (o, n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro o l.patients in gr~ o received i~ (an initial bolus dose of rg slowly intcavax~ rd mg as infusion over ~ rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj - rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, snd rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min -~a ~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than hour map < mmhg or use of vasoactive agents or blood lactates > mmol/ were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at . , , , hrs. an immunoradiometric assay was used to determine plasma concentrations of il (n< . ng/ml) and tnfc~ (n< pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n< . eu/ml)( endotoxine unit= pg). results : severe multiple trauma patients (age = _+ yrs, iss = -!-_ , saps = +'~, mean-+sd) were studied. they received + packed red cells during the first h. mean duration of collapsus before inclusion was . _+ . hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il level and saps was observed. in the early post-injury period phi, sao , svo , vo were significantly associated with ;il release (p< . at ho, h , h ). later a significant correlation existed between lactates and ii (h , h ). a peak of tnf was detected at and hrs. it was associated with low phi and low arterial ph of the early post-injury period (p< . iat ho, h , h ,h , h ) and with high lactate levels of later period (_>h ). only the late release of endotoxins (i{ ) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il in the early phase of trauma . i and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect brain wave monitor from january to june , . results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo ) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett ae ventilator with integrated metabolic monitor. inclusion criteria were; ) stable cardiorespirator s" status; ) systemic and pulmonary artery catheters already in situ; ) inspired oxygen < %. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, ug/kg, followed by an infusion of ug/kg/h to maintain the train-of-four ratio at . a further four sets of measured and calculated variables were obtained at min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo fick) , and measured oxygen consumption (vo gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than . and normal coronary circulation. for postoperation sedation propofol was administered in . mg/kg i.v. bolus, followed by a . mg/kgth perfusion. all data were registered before administering propofol and after minutes, the patients being hemodynamically stable and a rectal temperature of _+ . -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about years old, and the average period of aortic cross-clamp was . min. the adminstering of propofol caused a decrease in the coronary blood flow (- %), great curonary vein flow (- %), myocardial oxygen consumption (- %), regional myocardial oxygen constanption (- %), myocardial oxygen extraction (- %), regional myocardial ooxygen extraction (- %), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+ %) as well as in the great cardiac vein (+ %). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (- %), diastolic (- %) and mean (- %) arterial pressure, systemic vascular resistance (- %), and cardiac output (- %). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of . % "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than . and normal coronary anatomy, were selected. after the surgical operation, . "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of _+ . -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was -+ . years. during surgical operation the period of aortic cross-clamp was . _+ . rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (- %), coronary vascular resistance (- %), regional coronary vascular resistance (- %), regional myocardial oxygen consumption (- %), regional myocardial oxygen extraction (- %) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+ %) and in the great cardiac vein (+ %). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (- %), diastolic (- %), mean (- % ) arterial pressure, and systemic vascular resistances (- %) decreased, while cardiac output did not. discussion: the administering of . % "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: anaesthesias have been conducted in patients aged years ( females, males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of % insnffladon through mask. operations lasted from . - . h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao , t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics ( . mg/kg) and h -blockers ( . mg/kg) on the operational table was sumplemented by administration of . - . mg/kg of lidocaine, . . mkg/kg of clonidine, . - . mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: . - . mg/kg of droperidoi, .l- . mglkg of diazepam and analgesia: - mkg/kg of phentanyl, . -- . mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was - ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the -th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of - per rain. adequate external breathing: decrease (rr) by - per rain., with sao increase from % to - %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by -t % to the end of the operation with the initial moderate hyperglykhaemia of up to mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state ( - ) and analgesia lasted - b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt . /qtc . sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days and he received haloperidol, - mg/d (cumulative dose rag) for agitation and delirium. icu day : qt . /qtc . sec. icu day : for better control of delirium, trazodone " mg q hs was added. icu day : he developed frequent nonsustained ventdcular ectopy. icu day : qt . /qtc . sec, pha . , paco mm hg, pao mm hg, k . meq/l, mg . meq/l. later in icu day the patient had brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day : qt . /qtc . sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to mg/day. over past decade, cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental ( m), propofol ( p_g/ml), the solvent of propoful (intralipid), fentanyl ( ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm minutes, pmn minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp ( o - m) was measured in a two chamber well system for hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e ). % conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january to february , , data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of icus, of which enrolled at least one case. the total sample included patients. overall, . % of patients analyzed (t / ) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these patients in days in icu. although over drugs were reported to be used, pharmacological principles accounted alone for % of all prescriptions. opioids were actually used in % of prescriptions; propofol in % and benzodiazepine in . %. as regards the way of administration, intravenous administration was applied in % of cases and, followed by intramuscular in . %. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in % of patients and neuromuscular blockade agents (nmba) in %. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in , selected patients (pts) (> y, f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao > mmhg with flu < . ; d) stable ph> . with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (> .(i ml/kg/h); f) mediastinal bfeeding< ml/h for at least h; g) normothermia (core temp> ~ and no shivering). subsequent reintubation was for: ) rr> /min; ) spontancx)us ventilation for rain with paco > mmhg; ) pao < mmhg with fio > . ; ) ph> . ; ) heart rate>] bm; and/or ) non mental alertness; and ) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after h was considered: ) spontaneous breathing without any forrn of mechanical assistance; ) stability in haemodynamics; and ) elimination of fever threat. results: pts ( %) were extubated at h without complication; other pts ( %) at h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by h. only pts ( %) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation ( %= h and %= h), exempting pts with developed post-operative complications ( %=extubation< h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first - hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged - . group a ( patients) received baprenorphine hydrochloride (norphin) at dosages of #. • mg/kg. group b ( patients) received tramadel hydrochloride (t~aasl) st dosages of . z . mg/kg. before the procedure diazepam was administrated i.v. ( . ! . mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by . % on the onset of the procedure while sap and sv decreased by .%% and . %, respectively (p< . ). however, there were no reliable co chsnges. spoz ~educed by @. % (p< . ) and remained lower than the initial one after the procedure was oyez. when administrating tramsl min. after ste~ting the procedure sap and svr increased by ~ . % and . % respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o. ). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, , , ) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of up to microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by %. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate - rag/h) and antipyrine ( rag p.o.) were infused to ci with ( patients -group ) and without ( patientsgroup ) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group chypermetabolizer") as compared to group chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac ) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups ( pts a and pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl mg• kg < in ", pancuronium . mg x kg - and propofol mg • kg - in ". a blind investigator administered via another venous way at same speed ( ") saline in patients of group a and mg x kg - cac in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), ' after anesthesia induction (t ) and " after tracheal intubation (t ). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t . this trend was statistical significant (p < . ) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < . ) dramatically dropping at t , while in group b it showed a negligible decrease at t (p = ns) and value similar to baseline at t . svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was hours more than in control and made hours, though their average labor duration and in control was the same and made hours. % of the examined women experienced independant labor, as for the control group -only %. supplementary methods of anesthesia were required in % in control,that increased medicamentous depression of the fetus, but for the examined group -only in %. the examinees's newborns experienced favourable terminations, in control newborns were in state of light rate asphixia. analgetic effect was complete in control just in i %, and of examinees -in %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for % and in control only for %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of mg. than we were increased the dose to mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had -rhythm with slow and a-activity. an c~-activity index have been lower than %. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to %. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts ( - points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and min after each treatment. clonidine (i- ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of gg/mouse, tfl increased . -fold compared to time zero. l-arginine increased tfl at a high concentration (i gg/mouse). l-name suppressed tfl . -fold compared to time zero at a concentration of gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium ( , mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that , mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of female swine ( - kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n= ): induction with intravenous sodium thiopental in a dose of mg/kg, group b (n= ): propophol in a dose of mg/kg for induction'and mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with - it of cold ( oc) university of wisconsin solution for a period of hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c- , . mg/ml) was infused via portal vein and the liver was incubated at oc for rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (- oc). results: histology was completed in / experiments ( in group a and in group b). mononuclear infiltration (halothane type injury) was found in all specimens ( / ). focal zonal necrosis and acidophilic bodies were found in specimens from group a ( / and / respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b ( / ). smears of hepatocytes -beth formalin fixed and cryopreserved at - oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: patients were treated from jan. till dec. results: in patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic ( ml . ~ bupivacaine) when analgesia was first demanded; second group received ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature < ~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, ; i : - objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in during a period of months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of . % on the short stay unit and . % on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: ) there is no indication of a reduction in patient days with pressure sores (short stay unit: %, long stay unit: %); ) there is a reduction in severity of pressure sores (grade iv decreased from . % to . %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from . to . and the mean frequency of nursing patients on alternate sides decreased from . to . times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads ( - times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' x with the so called bookend method and are connected to a low vacuum suction system ( - kph) results: we studied the frequency of dressings changes on patients in the itu. in total they had days with an open abdomen. during this period there were dressing changes. this means one dressing change every days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo c: siemens), ventilation modes ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (- cm h ) was produced with a tv of ml and a frequency of /min in the ippv mode with trigger level on - cm h and a peep level of + cm h . the servo c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set ( cm h ), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( - cm h ) and the ippv mode the largest ( - cm h ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q , n = , items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by persons ( %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q , n = , items). at the issue, iger was created. results : q : responders ( %). law's misinformation : to %, disagreement for limitation of intensive cares : %, hope to improve collective decisions and creation of iger : % q : responders ( %) : satisfied by the training sessions : %, non satisfied : %, want to continue the project : %, refuse : %. iger was constitute by physicians, nurses, secretary, dietetic (n = ). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: ) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. ) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the st, nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in patients ( hmef and hme) h and h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, ( %) ventilator tubing yielded microorganisms in, at least, one sample as compared to ( %) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups ( % vs %, p:ns, for hmef and hme respectively). among the bacterial species isolated from ventilator tubing in the hmef group, ( %) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group ( / , %). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air ( % • vs % • .%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after h of mechanical ventilation in the hme group as compared to hme group ( . % • vs . % • %; p= . ). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in ~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in ~ end endocrine pancreatic tumors in %. in the intensive oere unit, these patients wore screened by noninwsive monitoring in ~ of cases: and invasive monitoring was applied in % of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of patient submitted to wchf during the last year. we used a total of filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x test to compare the results. results" from nurses of staff refused to flu the quetionary, and were not available. of the remaining, %were v~men and % men. the mean age were . % had an svable contract and ( eventual, the mean professional experience were of years and % worked in the unit since more than years. the % answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: ) to inform about patient mood. ) to inform about happenings from the last visit. ) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients ( male and female, aged + , and , , years respectively, ~+sym) being on therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without . during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa and sa were signif~canthy decreased in the case of oxygen discontinuation (p< , i). paco was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p< , ) . heart rate increased in all phases of the transport when administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s ~ment depression was progressively increasing and became very high at the end of transport in the case of therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao and sa , increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results: patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was , +- , . the duration of coma was from rain. to hour,average , +- ,sh.ln the group (the deads) the depth of come was , +- , .the artificial lung ventilation was used in all patients:in the group , +- , days,in the ~ , +- , days.apallish syndrome developed in cases,in patients diagnozed <<brain death~. the th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( group) and slow ( group) activity. the relative power of delta-and theta-ranges was about - per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the group decreased on , % and , z, in the -on , % and (; , %.general peripheral resistance (gpr) increased in the group on , %,in the -on (; , %.in the group brain blood flow was increased, in the -decreased on , n.in the group there was the normotonic type of reg-curve,in the -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on and n) and vascular ones (an increase of serotonin concentration on and %, hi stamine content on and %) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the group on %,in the - %.the level of middle molecules (mm) increased in the group on , %, in the -on , %,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was , . immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: . protective inhibition of brain and reduction of its energy requirements. . recovery of cell and vascular membrane fuoctions. .recovery of blood circulatiom .oxygenation of nervous tissue and drainage of brain disintegration products.in patientshbo carried out. .active methods of detoxication (homo-and enterosorption). .correction of cerebral metabolism. . dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. ( ). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co ( ), asystolia ( ), eclampsia ( ), acute renal failure ( ), control group ( ). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in monopolar channels. the estimation of eeg patterns by zhirmunskaya method ( ) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) , , , hz. we studied also the figures of absolute and relative power in such diapason : delta ( - hz), theta ( - hz), alpha ( - hz), betal( - hz), beta ( hz and more). results: all eeo changes were divided into following types: -organized ( , groups); iii -asynehronic ( group); iv -disorganized with prevalence of alpha-waves ( , , groups); v -disorganized with prevalence of delta-and theta activity ( , , ; , groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf- ,with effective area- cm,the volume of extracorporal contour- ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. ( ). post-operative cerebral neoplasm ( ), post-operative subdural hematoma ( ). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the minutes p st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) ( ) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: patients were included in this study: men and women. the mean age was years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was h. m. +/- h. m. hypertension was found in patients ( %), ischemic heart disease in patients ( %), hypercholesterolemia in patients ( %), hypertrigliceridemia in patients ( %), smoking in patients ( %), atrial fibrillation in patient ( %), history of epilepsy in patient ( %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in patients ( %), a single hypodense lesion in patients ( %). in patients electroencephalogram was normal ( %), in patients there were widespread nonspecific electrical changes ( %), in patients there were focal nonspecific eeg abnormalities ( %). conclusion: in our study tga was more common in women ( %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. march thirty-seven patients with intracranial aneurysms were operated on within hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n= ) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in ~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_. bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin ~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: ) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, )the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and ) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. ; : - ) . we wondered whether co vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: patients ( m/ f, age - yrs, glasgow - ) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco , , , and mmhg by increasing pico with mechanical ventilation kept constant. normal co vasoreactivity was defined as an increase in cbf of at least i ml/min. g per mmhg paco . results: patients had normal co vasoreactivity bilaterally, patients had altered co vasoreactivity at both sides, and patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the patients left cbf was in mean ! ml/min. g lower than right cbf (figure methods: following institutional approval piglets (body weight :tl . ) were anaesthetized by % fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs- abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at mmhg was achieved by appropriate balloon inflation for rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio =l on respirator (o). samples were obtained at time ( ), ' min at hypoperfusion ( ) arid at reperfijsion at ' ( ), ' ( ) and ' ( ). pao , pjo and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of ~tm hydrogen peroxide (h ). resul'~s: the mean pao was mmt-ig for group i and methods: we retrospectively reviewed all upper gi-endoscopies, performed in the period january -july in patients ( men and women) admitted at the icu's of our hospital. results: it concerned surgical, medical, eardiological and neurological patients with a mean age of . yrs (range: - ). in %, the endoscopy was performed at the icu and in % at the endoscopy department. in % of the cases, the endoscopy was primarily diagnostic, of which % was performed for localization of upper gi blood loss. in % the endoscopy was primarily thempentic, of which % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees ( %), duodenal ulcer ( %), oesophagitis ( %), gastric ulcer ( %), others ( %) and none ( %). as coincidental findings were noted: cesophagitis ( %), gastritis ( %), gastric deer ( %), duodenal ulcer ( %), duodenitis ( %), oesophageal ulcer ( %) and others ( %). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis ( ). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above mmoltl, measurement was repeated every hours for hours or until normaiisation (blood lactate level less than mmol/ ). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was , % and mortafity was , % in a group of patients with blood lactate level less than , mmol/l (n = ). frequency of complications ( , %) was significantly increased in a group of patients with blood lactate levels , - mmol/l (n = ), mortality was , %. mortality ( %) and frequency of complications ( %) were significantly increased in a group of patients with blood lactate levels above mmol/l (n = ). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo ) after major liver surgery, as shvo is considered an indirect parameter for splanchthc and hepatic blood flow. methods: consecutive patients were included in this study after liver resections for primary or secondary liver tumors. patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in out of attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein ( . _+ % rain vs. . + . rain; p < . ). the procedure requires a total of between and minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo showed a span from . % to . % with a mean of . % -+ . %. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without ( . % • . % vs. . % • . %; p < . ). (b) a negative correlation between shvo immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = - . ; p < . ). this correlation could also be seen for the first post-operative hours (r = - . ; p < . ). (c) a negative correlation between shvo and the difference between arterial and hepatic venous lactate levels was found (r = - . ; p < . ). conclusions: the routine measurement of shvo appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo findings cannot be given yet. methods: in cases after major liver resection, in which abnormally low readings of shvo suggested an impaired hepatic blood flow, pgi was applied at a dose rate of ng/kg/min. as shvo can be considered an indirect parameter for hepatic blood flow, the effect of pgi infusion on shvo was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo for all patients .was . % ( - - - ). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi lead to a sustained increase of shvo z to an average of . % ( . - , ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi infusion could be observed. both patients died and days respectively after operation in protracted liver failure. side effects of pgi included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to % as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h -receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/ could have hidden smaller differences between c and r as has been described in volunteers. between april , and april , , patients with severe acute pancreatitis were admitted to participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ ) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and were assigned to the control group. there were deaths in the control group ( %), compared with deaths ( %) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = . ). this difference was mainly caused by a reduction of late mortality (> weeks) due to significant reduction of gram-negative panreatic infection (p = . ). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < . ). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients ( %) and transient gramnegative pancreatic infection was seen in one ( %). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p- . active c provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline ( , - , % naci solution) in electrochemical set e~io- (russin,moscow). methods: . the nacio in concentration ragfl ( - ml/ h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis - /t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. . after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i mg/t in : ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca g/l), the concanlration of medium molecular oligopeptides (mm , ) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: . the intravenous administration of nac excels detoxicative effect of hemosortion by - % provides effictive presentation of protein components and blood cells and improves the transport function of albumin by %. . the return of exfused plasma after its purification ieco was - %. only the remaning - % of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of mos pts, m and f, mean age + . yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin days (mean . ). the mean peak level of ast, alt and ldh was iu/l (range to ), iu/l (range to ) and iu/l (range to ) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of . mg/dl (range . to . ), while altered coagulation paran-,ete's (pt> . times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt ( . %). renal impairment (cr> . mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure ( %) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range - pg/kg/min) and dopamine (range - pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired ( . %), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure ( %). it is concluded that: ) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. ) clinically significant coagulopathy is uncommon complication of ih. ) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, , and hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: pts, ~( , %), ( , %) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age: , • years. hean stay in icu: , • days. pts were operated, of them twice. hean value of ranson's scale: , • ( - ). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf - pts. b)early ards with typical xrf - pts( died), c)early arf with xrf(atelectasis,infiltrates)- pts( died). d)late ards with typical xrf- pts( died), e)pleural effusions in various combinations with the above patterns - pts. overall mortality rate: / = , %. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. )even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. )deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. )delay of surgical therapy is related to worse prognosis(p<o, ) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o, ). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of months all surgical patients on icu were registrated prospectively (n = ). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. , m!ll. of single patient informatioas. results: of patients didn't suffer from liver disease or have any operation on liver or bile tract. of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by patients. at a ,,cut-off" of rag% (total-bilirubine) the sensitivity was , , specivity , , positive predicive level , , negative predictive level , for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface . cm z, flow= itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight gr (min , max gr) were obtained and after a mean cold ischemia time of min were perfused fo} a mean of . h (min . , max h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of ( - ) mmhg at ~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t ) sampling: po , " + + " pco , ph, hco -, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from . at t o to . at t while mean output po fell from at t o to at t . mean output ast values increased from at t o to > at t while mean output alp values increased from . at t o to at t . mean output k + values increased from . at t o to > at t . histology revealed lesions of ischemic necrosis, more prominent after t . conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between and the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years organs were donated from potential donors i single, multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from ( ) ,to ( ) with a concomitant drastic reduction of donations from % in to % in ; amounting to a net unchanged number of donations over the last years ( = ; = ) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from % in to % in despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho ) provides online information about hepatic-splanchnic oxygen supply-demand ratio [ ]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [ ] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi , flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho ) in human liver transplantation. methods: with institutional approval and informed consent, consecutive patients, mean age - -_ years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of . gg/kg/min and pgi at ng/kg/min were initiated for a rain period. each sequence was followed by baseline therapy for rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p< , . results: ng at . gg/kg/min induced a decrease of mean arterial pressure (map) ( _ [baseline] vs. + mmhg) and pulmonary artery wedge pressure (pcwp) ( j: [baseline] vs. : mmhg). cardiac index (ci) ( - vs. + l/rain/m ), oxygen delivery index (do i) ( -+ vs. + mgnfin) and svho ( _~ vs. -l-_ %) were decreased (p< . ). pgi at ng/kg/min induced a reduction in map ( • nm~. _g) and pcwp ( + mmhg). ci ( _+ l/rain/m ), do i ( : ml/min) and svhoz ( + %) were increased (!o< . ). vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. pgi increased systemic oxygen delivery in parallel with svho , suggesting a corresponding improvement of hepatic-splanchnic okygenation. thus, if vasedilator therapy is indicated in th orient receiving liver grafting, pgi appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, until now donors were involved into this prospective study. every other donor was treated with mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group -steroid pretreatment goup (y~= ), and group -control group (n= ). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the - th, th and th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group had slightly lower ereatinin level ( -+ g.,non vs. -+ gmol/ ) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group . the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august and september , patients with transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n= ) included microbiologic evidence and more than secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n= ) referred as control groups. results: -year patient survival was . % ( / ): patients died due to serious infections, while died for other reasons. mean bilimbin, stnf-rii-, ifn- -, il- -, il- -, il- -, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: _+ pg/ml vs • pg/ml; ifn- : _+ pg/ml vs . -+ . pg/ml; il- : -+ pg/ml vs -+ pg/ml; il- : -+ pg/ml vs _+ pg/ml; il- : _+ pg/ml vs • pg/ml; laminin: -+ ng/ml vs -+ ng/ml; neopterin: _+ nmol/ vs _+ nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: -+ mg/l vs • mg/ . conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: patients with transplants were monitored for various cytokines on a daily basis between august and september . rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, patients ( . %) developed rejection; patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further patients required additional treatment with fk or okt (steroid-resistant rejection). patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn- , il-ib, il- r, il- , il- , il- , hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: . -+ . mg/dl vs . -+ . rag/all; tnf-rii: -+ pg/ml vs _+ pg/ml; il- : +- pg/ml vs -+ pg/ml; neopterin _+ nmol/ vs -+ nmol/ ; hyaluronic acid: _+ ~tg/l vs _+ ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: _+ mg/l vs _+ mg/ . ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il- , hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals ( beds) between december and may . exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms and days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that % is critical value for cpv loss and for cev it is %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june and april patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to days (mean . days) we reported of these patients as possible organ recipients to eurotransplant. all of these patients were transplanted within hours, ( %) of them even within hours. the principal aetiologies were hepatitis b ( ), hepatitis c ( ), nanb hepatitis ( ), mushroom poisoning (amanita phalloides ). after transplantation patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was %, patients died within months after transplantation due to various reasons. patients were not referred for liver transplantation. of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning ( ), paracetamol intoxication ( ) and fulminant hepatitis a ( ). patients suffering from fulminant hepatitis ( ) or intoxication ( ) were excluded from emergency liver transplantation for various contraindications. of these patients ( %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within hours. but the good survival rate in the transplantation group ( %) the % recovery rate in the group, where there was no transplant-indication in our opinion and the fatal outcome ( % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t ), increased reverse t (rt ) with normal levels of tetraiodothyronine ( " ) and tsh, this lacking in " " levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: .to study thyroidal pattern in brain death patients potential organ donors. .to avoid organ impairment by administration of t . .to study the hemodynamic and hormonal changes after the administration of t in these patients. material and methods:population: brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group ) and unstable (group ). group received a bolus of . p.gr/kg. and a perfusion at a dose of - . p.gr]h of t . hormonal assays: total t (tt ), total " (tt ), tsh. fxee t (ft ), free " (ft ) and rt were determine at the moment of clinical brain death ( hrs) and in group two these assays were repeted at hours , and . results: patients ( male) with a mean age of years (range to yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were patients in group ( , %) and patients in group ( , %). hormonal pattern: at the moment of brain death tt was normal in cases ( , %) and decreased in i ( , %); tt was normal in patients ( , %) and decreased in ( , %); ft was normal in cases (i , %), decreased in ( , %); fl' was normal in patients ( , %) , decreased in ( , %) .rt was normal in cases ( , %) and increased in cases ( , %). there were no statistically significant differences in hormonal pattern between the two groups. only t levels at hours , and were significant in group . in the cases with ft decreased, the tt was normal in ( %) and decreased in ( %), tt was decreased in ( , %) and normal in ( , %), tsh was decreased in i ( , %), normal in ( , %) and increased in i( , %) and ft decreased in ( , %) and normal in ( , %) and rt was normal in ( , %) and increased in ( , %). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t . conclusions: . the hormonal pattern most often find in brain death patients was: normal tt , decreased tt , normal tsh, decreased ft , normal fr and normal rt . . there were discrepancies in the values of ft and tt . there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace ) could be detected by mra. methods: following institutional approval and informed consent, mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace : , , mmhg; arterial blood gas probes; avl). mra was performed with a . tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight- d-angiography (tr: ms; te: ms; fl: deg; slab: mm) for vessel diameter detection and a flash- d-gradient-echo-sequence (tr: ms; te: ms; fl: dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace correlated with changing signal intensities, whereby under hyperventilation a decrease of , % (p . ) and under hypoventilation an increase of . % (p . ) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of - . % (p _< . ) and in peripheral diameter of - . % (p _< , ). a change from normo-to hypoventilation produced an increase in proximal diameter of + . % (p < . ) and of + . % (p -< . ) in peripheral diameter. conclusions: pace related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co -reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: ) bronchoscopic potency of airways and air bronchogram sign } resolution time of atelectasis and broncoscopic potency of airways. ) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were , men women , seventeen patients had atelectasis of whole lung, of upper lobe, and of lower lobe. ten patients had atelectasis in right and in left lung. eight from patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [ from patients with air bronchogram sign ( %) and from without air bronchogram sign ( %), had bronchoscopic potency of airways, p> . ], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency , days and in bronchoscopically closed bronchi , days, p> , ). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign , days, and without air bronchogram sign , days. p> ). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer ( . mhz) and a linear transducer ( . mhz) using acuson xp/ c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. ) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. ) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. ) . from march to april we did examinations by cs in icu patients ( male, female; age from - ). patients examinations pulmonary embolism pneumonia pleural effusion us-guided thoracic punctions were performed in patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an % concordance. results of the tee classification were : class : n = ( %) ; class : n = ( %) ; class : n = ( %) ; class : n = ( %) class : n = ( %). therapeutic implications of tee in class patients were : cardiac surgery in patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in patients with endocarditis and prescription of anticoagulant therapy in patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in patients admitted to the icu, an ultrasound-guided method (ultrasound group : patients) with an external landmark guided technique (control group : patients). all jugular vein accesses were performed by young interns with an experience of < procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in patients ( p.cent) in the control group (p < . ). average access time was longer in the control group ( • sec. vs • see. ; p = . ) and puncture of the carotid artery occurred in patients in each group (p = . ). patients ( p.cent) in the ultrasound group and patients ( p.cent) ia the control group (p < . ) were cannulated in rain. or less. the cannula was therefore unabie to be inserted within minutes in patients in the control group, with failure of eannulation in of these patients ( p.cent). failure was due to thrombosis (n = ), small calibre of the internal jugular vein (< ram) (n = ), abnormal vascular relations (n = ) or cervical irridation (n = ). among the primary failures of cannulation, an internal jugular vein catheter was able to be inserted in cases by an experienced physician on the side initially selected and with ultrasound guidance in cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining cases. jugular cannulation was obtained at the first attempt in p.cent in the control group and p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of - %. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from - %. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases ( male, i female, - years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in pts, a massive pe in pts. one patient (pt) had a cardiac arrest. in all cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in cases. all pts were treated with mg rt-pa, pts in a front-loaded regimen over minutes, pt over minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after to hours. all pts survived and were put on coumadine, pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein - cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a . mhz transducer (ultrasound depth - cm) fitted with a needle guide and a cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in patients catheters were introduced.in cases more than one ( - ) attempt was made and in patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > - cm), during the procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of days (range - ) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first patients cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following patients duplex scanning performed after removal of the catheter yielded another cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation - cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary -bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from consecutive patients admitted to the icu from / / to / / were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over years evaluate the necessary efforts and expenses to implement a cis in the routine of a -bed stcu. methods: in june a commercially available, unix-based cis was installed on a -bed surgical icu. the goal was a paperless documentation at the bedside. after more than years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, - trained health care professionals for configuration, system administrator. on a single icu ( - beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo ). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab ; : ). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo ), at ips (sipso ) and at mid jugular vein ( th cervical vertebra) (smj ) bilaterally. results: we found significant differences between right sjbo and both right sipso (p= . ) and right smjo ( p= , ) and between left sjbo and both left sipso (p= . ) and left smjo (p= . ) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : channel electroencephalograph, -channel roencephalograph, ad-converter ( analog inputs, bit resolution, k hz), ibm dx , software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal . ), expert programs for estimation objective and humoral patient status (written in clipper . ) and statistics. there were used following programme-language instruments : borland c++ . , nantucket clipper . , ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: . direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. . indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner % of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to centers and we received responses ( . %). there was at least one uncological (i.e. with > % of cancer patients) icu in (% % an -year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at /minute and a temperature of ~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was /mm ~ with neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv ( %) (n - %) and cardiac index ( , ) (n , - ). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n= , - pmo/l) went from , to , after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: patients were included in a prospective study from june to november . patients presented arf defined by creatinin serum leve(s greater than pmol/l and previous normal levels. patients were divided in three groups. group i (control) : patients with arf who did not receive substitutive techniques. group ih patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii : patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was . and mean length of tcu stay was . days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics ( ) cardiovascular drugs ( ), gastrointestinal system drugs ( ), enteral and parenteral nutrition ( ), respiratory system drugs ( ), sedative, analgesics and paralysing agents ( ), parenteral solutions with electrolytes, vitamins and trace elements ( ), anti-inflammatory agents ( ), protein substitutes and immunomodulation agents ( ), anticoagulative agents ( ). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was . drs ($ ). total tc/patient was . drs ($ . . ). ii) partitioning total tc per group of drugs reveals : ( ) %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %. t ) concerning antibiotics which consist the major cost component, group a and group b contributed by . % and . % to the total icu tc respectively. group b were administered to . % of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. -to determinate the variations of the rso during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso ) and rso were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam ( mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental ( mg/kg),fentanyl ( mcgr/kg) and atracnrium ( , mg/kg) have been used for induction of anesthesia. co te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane ( , - , %) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each minutes: blood pressure, cardiac frequency, pso , co te and rso . the rso cerebral variate in relation with: the anesthetic induction, blood ~ressure, co te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso cerebral was in relation with the :ross-clampping carotid ( minimal value: ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than anesthesia in chelyabinsk emergency hospital every year. to % patients of it emergency anesthesia is applied. more than patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than % of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of parameters simultaneously. results: pathients extremly needed peryoperative monitoring of hemodinamics. from these patients more % had stroke volume (sv) less than ml, n -co less than . /mim/m , % -ejection fraction (ef) less than n and % -puls bioimpedans microvessels (pbm) less than morn. patient had intensive care in special department. out of died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do ), oxygen consumption (vo ), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do ]. methods: patients ( males and females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo , do , ci, (a-v)do were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a -hour study period. a total number of measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p< . value was considered as significant. results: mean values were phi= . + . , do = + , vo = + , c. = . + . , (a-v)do = . + . . no correlation was found between phi and do , phi and vo , phi and c.i, phi and (a-v)do . on the contrary in patients phi remained below . for more than hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high ( %). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, berlingen) in combination with a standard gastroscope. measurements were performed on ventilated, traumatized patients (ages - years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of cm was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~ ) and standard p~ (ps st) consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in patients (mean age -* y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at ~ c using the abl radiometer for po , pco: and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the p~ at ph= . , pco:= mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps "in vivo" (i.e. the pso at the patient's value of ph, pco and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was . -* . houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo and oxygen extraction ratio (oer), r= . ,p= . . the improvement of ci ( . -* . to . + . l/min/m , p< . ) induced a significant increase in svo~ ( . -* . to . • . %, p<. ). a significant decrease in p ( . • . to . • . mmhg, p< . ) without any significant change in p~ st ( . • . to . • . mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: ( )personal data ( )patient's pathology ( ) laboratory and~ monitor lug data ( )drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. ( )reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month ( ) access to de questionnaired apache ii to load up new data. ( ) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: ( )detailed drugs interactions, including toxic elements ( )diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy ( ) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and ( )perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = days, los = - days, los = - da~, los = - days, los > day~. area under the carve (auc) was ud~ninted for each ro~. s~ing: imlimlcus. patents: of ~ pts comec~ively admired ~ a period of three yeet~ ( ) ( ) ( ) , a total of was i~leded in this study. pts without saps, p~ yolmger them yearn, p~ with los shorter ~ hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g : . , p > . ; auc = . i- . ) and in validation ~t (g.o.g g : . , p > . ; auc = . ..+ . ). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being . i- . in pts with los = days el . ~. ia tm with los > da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: .-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with items grouped in sub-scales: physiological functions ( items); functional capacity ( items) and subjective aspects ( items). qol is classified in levels: normality ( points); slight deterioration ( - points);moderate deterioration ( - points); significant deterioration (>i points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first hours as evaluated by tiss, and related factors. results: the sample was , patients, sge . ~ . years. severity by apache ii system was . • points. the tiss score was . • points, distributed as follows: i (<i points): %; ii (i - points): %; iii ( - points): %; iv (> points): %.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = . , p < . ), and a very weak but negative correlation between tiss and age (r = - . , p < . ), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v . for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of items classified in categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances ( . ), b) all generic names of medications available in our country for each substance, c) incompatibilities ( . cases) and d) interactions with other substances ( . cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on consexa~e ~ilically ill paliffas, subdivided in series following the diastases at the admission: medical pafiea~ ( s and ns), surgical patients ( s and ns), a~d poliwauntas ( s and ns). follow up was done at d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at fin~es (t): at fiae ~draission (t ), at x~ars from t (t ), at (f ), (y ), (t ), % (t ) and horus from t cf ). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < . w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t has a sensitivi .ly of ~ ,and a ~ecificity of . %: sv > . nffmin/n~, map> mmhg, pmap< nmalqg cvp<i nunhg will nmah& lvswi> g m/m , sxo > ~ do > mlhnin/m , o er< %. for lx~dical l~tienls at t the following criteric~a has a ser~tivi.ty of % and a ~zificity of . ~ cvp< . mn~g, sao > %, s,g) > ~ vo i< ml/nfin/m , o er< %, shunt< % survlvops' data of the series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n [ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh ~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure ) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied patients ventilated with a siemens c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of inflations ( , ,... ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves (( , c , c the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of paired act/p't-/" values was analysed in patients ( m., f., + y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of . u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within minutes. results ac-i -~ pi-f~ cact/~pi r = , ) cvc samples + + . v.femoralis samples " + + p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january through august , we studied filter surviva[ and hemorrhagic complications during filter periods in critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: filters ( %) were disconnected because of coagulation. mean survival of multiflow an filters was twofold shorter compared to survival of fh gambm filters. a total of hemorrhagic complications occurred of which three patients died at aptt values of respectively , and seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased % (relative risk . , %c . - . ) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased % (relative risk . , %ci . - . ) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between and seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure mmhg on admission and hours later were respectively . + . mmhg, . +_ . mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and hours later were respectively, pas; . _+ . , + . mmhg, ad; . _+ . , . _+ . ng/ml, nor; . _+ . , . + . ng/ml, pra; . _+ . , . _+ . ng/ml/hr. the ev values correlated significantly with ad (r= . , p= . , n= ), nor (r= . , p= . , n= ), pra (r= . , p= . , n= ). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p= . ) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf , , m , fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s- . the reaction is allowed to proceed at,. room temperature for minutes before discontinuation. the absorbance at nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied patients, who were admitted to our neurosurgical intensive care unit between january and march with head injury and no other associated pathology (age - yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) - hours after trauma (t ) and t hours later (t the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd( ).tbe n'mimum effect occurs after hours from the fast administration and is still present after hours. ~- patients, mean age --- yrs., weight --- kg were studied. ( . %) were male and ( . %) female. onderwent hip prosthesis ( previously plate and screw removal) hip revim'un ( stem, cop and stem + cop), tutal knee prosthesis, in the st anaesthesidogy depl from - to - - . as for antithromboembolic ptephylam, apart from hemodihitiun pts were with treated indobufen ndo), with heparin ealdum caeca) and with low mo!lecular weight hepam (lwr, ). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p= . l) in the padeats treated with indobufen ( . ) compared .'ith heca ( . %). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p= . l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro . ) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec, .a ( . -+ . days) and lmwh ( . +- a days) compared with indo(ll. _+ a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from - a + mmhg, which activates a peristaltic pump connected m drainage robes. the bt recovery display shows hourly bleeding in the first hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at - mmhg and then modified according to bleeding/minute, g bt recovery also has an alarm that sounds automatically if.' blood loss is more than ml/hour; air is in the circuit; the batteries are running low. materials and methods: pts were studied ( m and ~), aged . -+ .lyears, basal hemoglobin . -+ (range . - . )g/all, treated from st january, to mst december, in the st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision ( pts), cup revision (~ipts), stem revision ( pts), total knee revision ( pts). the average dumtion of the operations was -+ min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on pts. and integrated (epidural analgesia + light general) on the remaining t . anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in ( . %)pts., calalc heparin in ( . %)pts., low molecular weight heparin in ( . %)pts.; pt did not give a predepoalt of blood, gave unit, pts units, pts units, pts units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was -+ ml, average postop salvage was -+ mi the average intra+postop +- ml. average postop loss was -+ ml. the global incidence of postop complications was: h~natomata . %, dvt . %, pulmonary thromboembolism , , myocardiac ischemia . %, acute myocardic infarction . %, respiratory deflciecy . %, arrhythmia %, cystitis . % there were nn complications in . % of pts. postop bleeding over ml in under minutes (with bleeding alarm activation) occurred in pts ( . %). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p= . ) and with a significant decrease (p~ . ) in the pruthrombic activity detected about hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in % of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references )borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. patients with sah were studied. in of them sah was due to closed eraniocerebral injury and in the rema ining resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in patients with closed craniocerebral injury and in with vas. cular malformation despite the lack of clinical symptoms the tests in posttraumatic patients and in patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period ( - ) were reviewed. diagnosis of dic was based on the association of fibrinogen < g/ -platelets < / -fpd > ~tg/ml in the hours of the admission. results : patients -mean age + y -saps +_ -gestanional age _+ weeks -the two first conditions associated with d.i.c. were placental abruption ( %) and preeclampsia or eclampsia ( , %). bleeding episode was present in pts ( %) and surgical treatment has always been necessary. pts ( %) were given packed red ceils ( + u) and fresh frozen plasma ( + u). patients were given platelets packs. heparin was never administered. pts required mechanical ventilation and two patients hemodialysis. all the patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t h ~ % -f at t h , + , g/i). but platelets count remained low (plat. at t h + / ) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, patients with a dic score ( , japan) more than points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i- mg/kg/h for days or more) treated group and no foy group, each of patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, , and days after the admission. at-iii at units for days was administered if the at-iii at admission was less than %. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ %; group b, foy (+) and the at-iii < %" group c, foy (-) and the at-iii %; group d, foy (~) anffthe at-iii < %, each of patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day to were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were %, %, % and % in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: adults, mean age years and children, mean age months ( me.- yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and / of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, , . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction , to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group (control} dogs and group (no inhaled) dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of and n~ (f[q , ) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation ( ppm) in group was started rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan ) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and , , and rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < , . : no produced an increase in arterial oxigenation (pao /fio~ ratio) and reduced pap before pe induction in group . after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to % ) spills automatically into the circle, balancing o uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. - ( %) underwent conventional surgical therapeutics. " ( %) with resection of tracheal stenosis with end-to-end anastomosis(rts). i ( %) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. - ( , %) have received endoscopic laser ablation with or without calibration tubes. of them ( , %) are receiving continued endotracheal treatment until now. ( , %) have sp wihout continued treatment. -i ( , %) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas ( % oxxygen, % carbon dioxide, % nitrogen); gaseous no ( ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, , , , , , , , and minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at + , % (percentage of baseline value, mean -+ sd) until min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after minutes ( , + , % vs _+ , %(p< . ); min. , -+ , %vs , _+ , %(p< . ); min. , _+ , % ( p < . ). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at , +_ , % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to , _+ , %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp- , cd p) and glycoproteiu (gp , lysosomal protein, cd ) are expressed only after platelet activation and degranulation. activation was quantified in thrombin ( . u/ml) and adp ( . ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n= ), who had no drugs for at least days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i ( . mm) ( -morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd p (fitcconjugated), and cd (fitc). only cd a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was . + . %, and increased to . _+ . % after thrembin-activation, and to . + . % in adp-stimulated samples. addition of sin- attenuated p-selectin expression to . - - % in thrombin (p<. , two-tailed paired t-test), and . + . % (p<. ) in adpactivated platelets. basal gp expression was . _+ . % and increased to . + . % in thrombin, and to . _+ . % in adp-stimulated samples. with sin-l, gp expression decreased to _+ . % (p<. ) in thrombin, and . : . (p . ) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died days after exposure. the metal he had been working with was a % cadmium alleation. blood cadmilam concentration days after exposure was . mcg cd/g cr, and urine cadmium concentration was . mcg/l. on postmortem examination, tissue cadmium concentrations were: blood ng/ml, liver ng/g, kidney ng/g and lung ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : patients were studied. on admission, plc ranged from . to mmol/l, and bcc from . to gmol/l. mean systolic blood pressure was • mm hg, mean arterial ph . • . , mean anion gap was . + . mmol/l and mean pao . • . kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = . ), anion gap (p = . ) and bcc (p = . ) but not with heart rate, pao , paco and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> retool/ was a sensitive and specific indicator of a blood cyanide concentration > ~tmol/ . sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a -year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were mmol/l and gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r= . , p< . ). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r = . ) with a blood half-life of . h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r = . ) with a blood half-life of . h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a g dose of hoco ( g in case of cardiovascular collapse) was administered intravenously over min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were females and males. the mean blood cn concentration was • pmol/ . the mean blood carbon monoxide was . • . mmol/ . nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < ~mol/ ), there was no significant change in arterial blood pressure. in the cn-intoxicated patients (blood cn > gmol/ ) a significant increase in blood pressure was observed both immediately (p < . ) and hour later (p < . ) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ ] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a -beduniversity hospital from through . the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between + and * + + • the best correlation with severity grade was found with qrs-and qtc-duration (p= . ), the tca-dose (p= . ) and hf (p= . ); tqrs did not correlate. patients died ( . %). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative , -methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken tablets of "eve" (mdea, approx. g) and g of "smack" (heroin) by oral route approximately h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was ng/ml (lethal range - ng/ml). trace amounts of cocaine and substantial amounts of heroin ( ngtml; mean value in heroin-related deaths: ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients ( females, male) with documented aip were seen over a -year period in the university hospital. patient was in clinical remission and were with the acute attack of aip, among them with respiratory paralysis were required artificial lung ventilation and -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range - mkg per/ kgr creatinine) fill from mkg on admission . mkg, then on - day raise to mkg and then during treatment with normosong and prasmapheresis lowest level was . mgk. fatalities occured in two females during attacks with proforma cerebral involvement and patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in anaesthetized and ventilated (fio . ) minipigs and dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above mmhg. results: flow ( and ppa matched pvz data (mean-+sem) are shown in the table. [zo = hz impedance (z; {dyn.sec_em- }); zl = first harmonic z; zc = characteristic z; z phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < . between dog and minipig, and before v~. after embolisation respectively]. before case report: a -yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt= ml, rr= /mm, fio =l, the patient had ph= . , pao = mmhg and paco = . systemic bp was / mmhg and hr= b/min with low dose epinephrine ( . g/kg/min) a thrombolytic infusion (rtpa: mg/ h) through a peripheral vein was started tee imaging and clinical status hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from / mmhg to / mmhg, etco increased from to mmhg and sao improved from % to % three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also ~, properties. case report.an obese },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine . /die was used for prophylaxis of thrombotic disease, on day th at . a.m. she started to be hypoxic (sa %), tach ,tardic l l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to r mm/hg, the central venous pressure (cvp) raised [rom lb to mm/hg and the end tidal co was mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase x i(i u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp / mm/hg, cvs mm/hg, spo %) despite a cominnus infusion of dobutamine fag/kg/min and adrenaline . ~tg/kg/min. at this stage a bolus of aoradrenaline ,g was given followed by a cnntinous infusion of . !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was / mmhg, the cvp mm/hg, the sao % and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i pte patients (pts), males (average age , yrs) and .q females (average age , yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in , % of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain ( %), dyspnea ( %), hemoptysis ( %), cough ( %) with association of two or more symptoms in %. chest xrays findings were abnormal in % with diaphragmal elevation ( , ~ lung opaeilies ( , %), atelectasis ( , %), plemal effusion ( , %), main pulmonary brancah asimetry ( , ~ oligemia ( %), heart shadow changes ( , %) and pulmonary arteries "cut off' ( , %). the association of two or more abnormalities was found in , % while normal chest x-rot was found in ~ of the cases. hypoxemia with pao < , kpa was found in , % followed with hypocapnia and respiratory alealosis in , % in , % of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i , %, ecg changes-st q t type in "~ , %. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet : , ) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared patients arrived in our intensive care unit (i.c.u.) in. : (group a) with arrived in : (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of , +/- , (hb) and +/- (hct) in group a while in group b mean value are , -/- , (hb) and +/- (hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took ml of patients' blood and separated it within min with rotation frequensy of /rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made - procedures during - days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from + . g/l to _+ . g/ and from + . % to _+ . % respectively). the time of blood coagulation by lee-white has increased by - . times (up to - rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from . + . ml/min to . _+ . ml/min (p < . ); b) the regional brain blood flow from . _+ . ml/min to . + . ml/min (p < . ); c) the brain blood flow in internal carotid arteries from . _+ . ml/min to . + . ml/min (p < . ). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of consecutive patients, on verapamil, on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil ( mg over minutes) was followed by a rapid loading infusion ( . mg/min) for minutes and finally a maintenance infusion ( . rag/rain) for hours. results: a mean plasma level of verapamil of ng/ml was obtained only after more than hours. atrial fibrillation occurred in five out of patients who tolerated the verapamil infusion, and in out of patients on placebo (p = . ). verapamil infusion was not tolerated in patients because of hypotension or a heart rate of less than /min, within hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion ( + ) compared to placebo ( + ). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to . + . g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to . _+ . g/l, monofibrin soluble complex concentration to . -+ . g/l, blood plasmin level was enhanced to . + . mmol/ , plasminogen proactivator level was also enhanced to . + . ram, plateletes aggregation has decreased to %. after plasmopheresis aggregation was decreased in . times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in . times, and plasminogtnt activator level in . times. at the same time we have observed increase in total antifibrinalitic activity of blood in . times. activity of activators plasmine and plasminogene proactivators has decreased in . times and in the same time activity of activation inhibitors and antiplasmines has increased in times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc ) monitoring based on thermodilution technique in critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique ( ml of ice-cold saline solution) was used to compare cc with intermittent bolus cardiac output (ic ) measurements. the following subgroups were prospectively studied: i. heart rate (hr) > beats/min, . cardiac output > i/min . cardiac output < . i/min, . rectal temperature > . ~ and . pa catheter was inserted for more than days. results: a total of pairs of ic and cc measurements were obtained from the patients. bias (ico measurement minus cc measurement) of all measurements were . • i/min and the % confidence limits (mean difference• were - . / . i/min. also in the subgroups, cc measurement agreed closely with ico measurement (c > i/min: bias= . • i/min; co < . i/min: bias=- . • i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc measurement with ic measurement neither (> ~ bias= . • i/min). conclusions: monitoring of cc using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january , this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since , patients aged to years (mean years) underwent cas without ecc. the mortality in programmed surgery was %. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: units per patient, b) need of mechanical ~entilation: i , hours, c) postoperative bleeding: cc, d) days at icui , . we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments per patient (p< , ), b) need of mechanical ventilation: hours (p< , ), c) postoperative bleeding: cc (p< , ), d) days at icu: (p< , ), e) programmed surgery mortality: % (p< , ). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography ( and l days after beginning of the treatment), stress-echocardiography and stress ecg ( - -th day after the onset of mi). enalapril was included into the treatment of pts with mi (study group), with normal or increased blood pressure, from the -st day of the disease. the data were compared with pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in pts of the study group and of control group, the arrhythmia episodes during stress test -in and pts and episodes of silent nocturnal isehemia -in and pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh % stress test. in pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by %; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef< %). besides cesf, the ejection fraction was calculated before and after administration of mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) . • . ef (simpson) . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! ! [ gedv, itbv and pdr showed a significant decrease dufing the first - h after the operation, cfi and rvef si~canfly improved after k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at h. comparison of cold/explorer m~ements sb wed good correlations. discussion: concerning m ~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least . ~ (max rain apartl the patients were separated in two groups according to their cardiac function: patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below % and patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction ( er), the q er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied mechanically ventilated icu patients ( • years) in stable hemodynamic status (no change in vasoactive treatment for at least hours), separated in groups: group = deep sedation, cardiac dysfunction required dobutamine (n= )r group = deep sedation (barbiturates), unaltered cardiac function (h=lo), group = moderate sedation, altered cardiac function (h= ) and group = moderate sedation, unaltered cardiac function (n= ). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline ( x) and twice (q. min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of hypertensive surgical patients. patients were allotted to groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)< . , patients; eukinetic (ef> . ),i patients and hyperkinetic (ef> . ),i patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p< . ) while systolic arterial pressure (sap) (p< . ) and peripheral resistance (pr) (p< . ) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p< . t) stroke volume (sv) (p< . l) and co (p< . ) were increased while pr(p< . t), sap(p< . ) and diastolic arterial pressure(p< . ) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p< . ) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p< . i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : palients, post cardiac surgery, uremic ( men, women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a fr polyurethane catheter with end and multiple side holes over ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes( - ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht= % ,protein , gr/dl) in all cases. in first hours the mean volume of fluid removed was ml ( to ml). in that period echo showed no residual fluid. the catheter remained within the pericardium to days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by -d echo guided perieardiocentesis. a -fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this -d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after rain, min, and finally with one hour rate, within first hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate hbenzoyl-phe-ala-pro (bpap; p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi( -m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as . xf v. results: similar values of %m ( . + . vs . • and v ( . • vs . • were observed in both and one lung respectively. a~k~ decreased from • ml/min (both ltmgs) to :~ (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the % reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients ( females), with a mean age of + (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin hours postoperatively. square wave stimuli of . msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding . msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, . • msec for the left nerve and . • mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients ( . • msec) , whereas left pnct was normal in patients ( . • msec) and abnormal in patients (incidence . %). in patients the left phrenic nerve was inexcitable and in patient left pnct was prolonged ( . msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction ( • vs • p= . ). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper to cm of lima were preserved (p= . ). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo shifts. methods: we studied males ( - yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature - c we set pump flow at . i.m~ .min - . when map was higher than mmhg we calculated vo by using fick equation. then we infused sodium nitropruaside (sn) to control map at - mmhg for min and we calculated vq . without changing the sn infusion rate we set q at . i.m' .min " . ten min later we measured vo . we took vo changes into consideration if greater than %. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo response to sn infusion during low and high q we classified pts in four groups (table). i. vo increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo increases with sn but the addition of high q results in systemic shunt. iii. vo increase during high q proves that vasodilatation can turn flow insufficient. iv. vo does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > . l/min/m , _< paop < mm hg,and svri __< mmhg/ll/min/m decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: aaar patients and pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > . sec or cpk-mb > %. results are presented as means _ -. sd. opt was achieved in of ( %) and in of ( %) in the pvs and aaar group, respectively. events did nat differ between groups of ( , %) and of ( , %) in the pvs and aaar group, respectively (p>o. ). mort was of ( %) and of ( . %) in the pvs and aaar group, respectively (p > . ), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, of ( %) patients who developed events in the pvs group had a ci < . in contrast to of ( %)in the aaar group. the positive and negative predictive value were % and % in the pvs and % and % in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; . pvs patients who have net achieved opt are at extraordinary risk of perioperative events; . preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< . ; ratio >_ . may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade ( ). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in consecutive patients (pts) after uncomplicated cardiac surgery ( coronary artery bypass grafts; valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : . _+ . /minlm~; laotate: + rag/i; cvp range : - rnmhg; pewp range : - mmhg. mean cvp/pcwp ratio before extubation is . ( % confidence imerval : . - . ) and after extubation, . ( % confidence interval : . -. . ), (ns, paired t-test). in % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = - . ) and after extubation (r = - . ) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = . ) and after extubation (r = - . ), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< . ) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as within a large range of cvp ( - mmhg) and pcwp ( - mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : ( ) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from to minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period june to december , patients with chest paros were treated within a eriod varying from minutes to hours from onset of symtoms. ages ranged from to (average , ), oelng males and females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l m.. potentia ami patients were then admitted to the coronary unit, [)atients, under age with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was minutes ( . ), -he total median delay was minutes ~ -i h. min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to -i minutes and treatment delays to less than i minutes results. following rmplementatlon of this protocol in january , fts were glven, with an over all average delay of minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a % reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly c agents to patients with suspect of ami from the begin!ng of september until the end of february . in this time there are two different periods, during the first months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from to ) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to - ~ c. renal dysfunction was defined as a % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x when appropriate. data are expressed as mean +__sd. p value less than . was considered statistically significant. results: thirtytwo patients ( , %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p< , ). mortality rate was statistically different in patients with or without renal dysfunction(~, , % and %, respectively, p= , ). incidence of acute renal failure following renal dysfunction was , % ( patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature ( ). however renal dysfunction appeared significantly associated with a poor prognosis. reference: -settergren g, ohqvist g current opinion in anaesthesiology , : - r ; , tzelepis, g. , , late complications were observed in % of cannulations: local infection in (i, %), catheter displacement by the patient in cases ( , %), catheter displacement during nursing care in ( , %) and malfunction in cases ( , %). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, selected patients ( _+ years) undergoing peripheral vascular surgery (n= ) or carotid endarterectomy (n= ) were investigated. patients included had either documented cad (n= ) or two or more (n= ) dsk factors (age > years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia > mg/dl). -lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and h, h, h, and h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression > . mv and/or new t inversion). in addition results: of the patients investigated developed ecg-documented pmi, % occurdng in the immediate postoperative phase. troponin i levels > . ng/ml were found in of these patients thus, comparing a cardiac troponin i cut-off level of ng/ml with intermittent -lead ecg recordings, we found a sensitivity of % and a specificity of % methods: demographic, clinical and ecg data were analyzed. . % of patients were male; . % female. cad was the most common underlying cardiac disease ( . %) and . % underwent open heart surgery. % received proeainamide for supraventricular and % for ven~cular arrhythmias. % received a loading dose. maintenance was provided by iv route in . % and by po in . % ( . %sr end . % ir). . % of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was • years, for group b • years, and for group c • years. a history of previous myocardial infarction was present in pts of group a, in of group b and in of group c. three pts in group a, in group b and in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was days ( - ) for group a we used a continuous fixed intravenous a infusion at a dose of the sn was % in groups a and b, % in c, and sp % for group a, (fixed defects included) and % for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, pts -group b, and pts -group c), transient hypotension ( pt -group c), headache ( pts, group c), dyspnea ( pt -group a), while st depression was seen in pts of group b and in pts in group c. the rate of a infusion was decreased to /kgr/min in one group b pt due to development of chest pain s five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch bertschat, e betbes blanch, l del nogal saez e -meneza nolla, j. nolla-salas pilz~ u puig de la bellacasa e scarpa, n. van de wetering objectives: only % of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines ( ) able to induce pronounced conduction block ( ). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted g catheters in gbs-and cidp -patients under sterile conditions. some of them had not responded very well to established therapies. - ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed ( - ml) over one week. results: the gbs patients improved after days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after days (median). patients not at all treated before ( / ) responded better than patients that had been pretreated ( / ) with plasmaexchange or intravenous immunoglobulines. / cidp patients drew benefit from treatment, stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower ( / ) than those for plasmaexchange or intravenous immunoglobulines. references:( )wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve ( ) . ( ) clinical observations were made in patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok- apparatus, the exposure within minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in bed university hospital, covering . inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march th , were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score > , at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant ( th, ~ and th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from patients ( apache ii score: . ) admitted in the icu, ( . %) enter the study protocol. seven ( , %) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was . vs . . the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, / and mof, / . conclusions: . -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. . -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(> years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, patients were evaluated; ( %)had pancreatitis of unknown etiology . results:( )patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity( % vs %),mortality( % vs %),and longer hospital stays than p~tierf~ with pancreatitis of known cause.( )the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.( )curlously,no difference existed in associated medical conditions between patierts withknown and ur ~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for > days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were . % ie at ph . _+ . ( % limits) and between old & new catheters were . %, ie ph j _+ . ( % limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis . therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of < . according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n= ); ih analgesiajsedation plus pirenzepine mg/day (n= ); .[ih anatgcsia/sedalkm plus sncraltate x [ g/day (n= ); iv: analgesidsedatkm plus pirenzcpine mghlay plus sucralfate x e/day (n= ). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p < . . / patients ( . %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb ( . + . vs . -+ . ). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = . ) has been shown. the frequency of gisb among the groups is as follows: h . %; lh . %; llh . %; iv: . % (ch -~ = . ; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" = . ; p= . ) and reached an overall-value of . %.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a -h esophageal ph-metry was performed in patients in recumbent position at ~ who had a ngt for more than days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than ) (ger-t) more than %, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale= - ) was noted. two ph-metrles repeated on h and on days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than (tph< ). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: out of patients presented a ger-t> %. eleven out of group i group (n= ) i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise ( , and w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: . +/- . i/min, vtc: - +/- ml and frc: . +/- . liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol ; : - . objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co vasoreactivity in brain injury (stroke ; : - ), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: mechanically ventilated patients (age - yrs, glasgow - ) in coma due to acute brain injury were investigated during stepwise changes in paco ( , , , and mmhg) by increasing inspired pco . middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of % n through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco . objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at mm hg. . ringer's solution, . . % gelatine solution, . % hydroxyaethylstarch (mean m.w. . ). we evaluated the following parameters within intervals of min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in consecutive patients ( sepsis/sirs, acute heart failure, ards, transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z- system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f - , df = , p < . , (figure) ). aitbv was correlated to ac in a linear fashion (r = . , f = , df = , p < .o ). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in anaesthetized pigs using a centrifugal pump with a f arterial catheter and a f left atrial aspiration line. animals were supported for two hours at a mean flow of . liter ( ' rpm), a mean hematocrit of % and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in patients with ph of various causes: primary; post atrial septal defect closure (asd); chronic thromboembolic (te); anorexigen; iv drugs; collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me . we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to control humans (mean %met = . % _+ . % s.d.), the mean %met in ph patients was . % _+ %. the %met in ph patients correlated inversely with cardiac output (r= . ), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph ( + ml/min) as compared to controls ( _+ ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from % to less than %. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in patients with (raaa).a short acth stimulation test (synacthen test; ug - acth iv) was performed at hrs within hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > . umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the patients investigated died and survived. mean cort basal in nonsurvivors was significantly (p< .o ) higher than in survivors; . (range . - . ) vs . (range . - , ). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; . (range . - . ) vs . (range . - . ). while patients showed a cort basal < . , no cort stim < . was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; vs and vs . conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: pts. aged + years, admitted to our ccu from to with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters ( f, cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into groups. in group a we included pts with anterior ami, in group b, pts with inferior ami, and in group c, pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p< . was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were + mmhg and _+ mmhg respectively. despite the signifanf correlation (p< . ) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, pts ( %) presented cvp> mrnhg and of these ( %) had pwp_> mmhg. in group , the cvp was _+ mmhg and the pwp, _+ mmhg. significant correlation (p< . ) between the two variables also existed, however it was impossible to predict the pwp value. pts ( %) had cvp> mmhg but only of these ( %) had pwp> mmhg, similar was the relation between cvp and pwp in group c (p< . ). cvp averaged + mmhg, and pwp, _+ mmhg. pts ( %) had cvp> mmhg and from these ( %) presented pwp> mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as - ~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated pts (age + yeats nyha class: ii, hi, iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo max determination, a -mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was . ( - ) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every hours until somatic death. we we detected severe diffuse hypokinesia (ef< %) in patients and mild hypokinesia in others (ef - %). systolic function was strictly normal in only patients. corrected qt interval (qtc) in ecg was . _+ . msec (normal range - msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref- ; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc ( + . + . , p < . ) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from . + . to . + . (p < . ); redv was reduced from . -+ to . _+ . (p < . ); resv was reduced from • . to + . (p < . ). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group ( . + . |• . ) compared with the c"group ( • . ($ . • . ) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: elective cardiac surgery patients. interventions: dobutamine was infused at ug/kg/min for hours immediately after admission to the icu. hemodynamics were measured every minute periods until hours and again hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied patients . -+ t . years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< mmhg, urine output< ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the th post infarction day and months later. results: three patients refused iabp treatment and / survived on the th day. on the th day / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p < . ). on the th month / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p< . ). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from to ; females and males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n= ), valvular replacement (vr, n= ), combination of both (vr-cabg, n= ), or others (n= ) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x with continuity correction when appropriate. results: seventy hree patients ( . %) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p< . ). type of surgery, preoperative ef, and nyha classification are the first significant factors related to inotropic support (p< . ). most patients operated on for double-vr or vr=cabg required inotropic support ( and %, respectively). postoperative mortality was higher in patients receiving inotropic support ( , % vs , % 'overall mortality, p= . ). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every - seconds. cardiac output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa- sing do i to at least ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached mmhg. vasoactive or inotropic drugs were infused if the desired do was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics . materials: asa group ill-iv patients with a mean age of years (range - ) and a mean weight of kg (range - )) scheduled for major abdominal surgery were included. results: patients were excluded because do i could not be raised at all. mean do i was increased from ml/min/m (range - ) to ml/min/m (range - ). mean volume of preoperativdy infused colloid was ml (range - ). during surgery ml (range ) of colloid was infused. mean length of surgery was minutes (range - ). mean blood loss was ml (range ). expected mortality and morbidity rates ("possum") were % and %, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of % and %, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in patients (pts) with severe left heart faille (left veutrieul x filling pressure (lvfp) > mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in furosemide (f) + d butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n= ): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am ) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: consecutive patiens with ami, from october , to february , , admitted within hours alter onset of symptoms, undergone successfull systemic thrombolysis. of them were anterior (group a) and inferior (group b) . noradrenaline plasma levels at (na ), (na ) and (na ) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns . ns means -sem (normal limit for our laboratory: na < / pg/ml; ck < u/i ) conclusions: . the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. . the decrease in na is more evidence in amis with anterior location. . this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. . there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: patients scheduled for multiple cardiac valve replacement in were divided into two groups: group i ( patients) -without pp; group ii ( patients) -pp was performed preoperatively. the first pp was made ten days and the second - days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing ( - ~ and absence of dmso. this made it possible to preserve % functionally active platelets during days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the st p. o. d. was significantly greatest in the group i ( _+ ml) and all the patients required transfusion of the donor blood ( + ml) whereas the blood loss in group ii was +_ ml and olny patients required the donor blood. the number of platelets on the st p.o.d, was _+ . /l (group i) and + . /l (group ii), p < . .conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the patients of the swiss centers) and in the patients randomized in the isis- trail mortality rate in women was . % ( / ) compared to . % ( / ) in men; in switzerland: in-hospital mortality for women was . % ( / ), for men . % ( / ).the table shows the results of isis- in terms of odds ratios and their % confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. ( ) and bubble ( ) oxygenators a, ere used. anaesthesia was balanced and pts were extubated to hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia ( ), after induction of anaesthesia (i[), mins after cpb (iii) (iv) and (v) hrs afiter cpb, rains after extubation (vi), hrs after cpb (v[ ) and on the nd, rd, th, th and tb postoperative day (pd) (viii, x, x, xi, xi , respectively). analysis of data was performed by two-way analysis of variance, p < . being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the th pd. qs/qt reached maximum at rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over ubbie type oxygenators. the aim of this study was to evaluate the influence of x 'genator type on pulmonary shunt (as/at) after coronary surgery. methods: patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a (n = ) with a membrane o~genator and a (n = ) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), mins after extubation ( ), hrs alter cpb ( ) and on the nd, rd, th, th and th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < . being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at rains after extubation (ii) in a group. the difl'ereuce between the two groups was no more significant from hrs after cpb (iii) to the end of the investigated period. ! i * p < a. s betw~n ~o~ conclusions: membrane ox 'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation ( to hrs after cpb). the difference in qs/qt disappeared his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence ( to % ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent ( - % are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in patients on admission to the icu, using a nova electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs ). normal range of img++ was found between . - . mmot/l (healthy volunteers). results: for the entire population, we found a % prevalence ( / ) of hypomagnesemia (figure ) . among the surgical patients, the prevalence was highest after cardiac surgery ( %) and after thoracic surgery ( %) and was lowest after neurosurgery ( %). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure ( % for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i- %. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from / to / consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was % ( ). in all cases diagnosis was proven by a positive hipa. / patients died. in / hatt could be confirmed before severe thromboembolic complications occured. / patients developed a deep vein thrombosis (dvt), / dvt and pulmonary embolism (pe), / dvt, pe and arterial thrombosis (at) and / a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of pts. is %. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality ( / ). computed tom graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of patients with cardiogenic shock, as defined by an initial ct below . l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n= ), heart failure (n= ), valvulopathy (n= ) or recent cardiac surgery (n= ). after exclusion of patients with concurrently suspected/documented infection, this study included patients, of whom ( . %) survived. treatment of shock included dopamine (n= ), dobutamine (n= ), norepinephrine (n= ) and epinephrine (n= ). patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about - mm hg ayter single drug administration observed only in patients. ezap monotherapy accomplished during one week with mg daily dose. hypotensive effect observed in patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of mg. this new drug combination permits to lower ap in patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra( , %)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in % patients with ah and low pra. methods: in patients with cardiogenic shock due to ischemic heart disease (n= ), heart failure (n= ) and valvulopathy (n= ), hemod aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n= ), dopamine (n= ), norepinephrine (n=i ) and epinephrine (n= objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing - kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h it of heparinised r/l solution at ~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing - kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was ml. temperature was maintained at ~ and portal vein pressure at ( - ) mmhg. the flow was . - . ml/gr of graft liver mass per minute. observation period was hours (t ). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t = mmhg , t = mmhg), hr (t = , t = ), rap (t = mmhg , t = mmhg), pap (t = mmhg, t = mmhg), pcwp (t = mmhg, t = ~mhg), svr (t = dyn'sec/cm ' , t = dyn'seclcm~ pvr (t = dyn.sec/cm o, t = dyn.sec/cm ,'~), co (t = . t/min, t = . t/min), do (t = ml/min, t = . ml/min), vo (t = ml/min, t = ml/min), o er (t = . %, t = . % ), ph (to= . , t = . ), po (t = mmhg, t = mmhg), pco (t = mmhg, t = mmhg), pvo (t = mmhg, t = mmhg), svo (t = %, t = %), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. consumption, co production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: paced pts (mean age +- yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to . ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity %), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a -year-old woman presented a year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was mm hg, pulse beats/mir~ute, hematocrit . , seln~n protein /i, and se~um albumin q/l. an leg-kappa pa[apfotein was demostrated ( . g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, . units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in critically ill patients men and women ages from - who had sepsis -arf congestive heart failure postoperative multiple organ failure and polytrauma .this method was applied to these patients from to hours. % of the patients recovered completely their kidney function, % improved their kidney function and % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to groups: in group i, (n= ) airf was inducted by bilateral renal artery clamping for rain. group h (n= ) rats underwent a sham procedure. in each group, rats were dialyzed twice ( th and th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an ) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): . _+ . ; cupro: . _+ . ; hemo: . _+ . ; an : . _+ . ). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day (p< . ), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: patients who underwent first cadaver kidney transplantation in our unit between january and december in were involved. the recipients were divided into groups: group i." non functioning graft (n= ); group ii: delayed graft function (n= ), group ili: good graft function (n= ). the grouping criteria were: a/haemodialysis in the fii~t postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. ( . ) and croup ii. and iii. ( . and . , p< . ). intmo! emtive significant differences were found between the different groups in systolic blood pressure (group i. hgrmn, group ii. hgnnn, group iii. hgmm, p< . ), mean arterial pressure (group i. hgmm, vs. group ii. hgnun p< . , vs. group iii. hgmm p< . ), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. inin. vs. group ii. rain. p< . , vs. group i. rain. p< . !). the rejection rate was higher in the first days in the patients with non-functioning grafts (group i. % and group ii. % vs. group iii. %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp . key: cord- -atbjwpo authors: nan title: poster sessions date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: atbjwpo nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v epitope tag into the n-terminal of klf gene. insertion into the target side of klf gene via crispr-cas system provided an opportunity to overwhelm the above mentioned obstacles. v epitope tag would not interfere with the function of the klf and also enable us to recognize endogenous klf via anti-v antibody in the mouse myoblast cell lines (c c ). we confirmed the targeted insertion into the exon of the klf gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc , but not ctf is detached from chromatin. our data also shows that meanwhile, rad checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc and ctf are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc has a more specific role in the process of adaptation. our data demonstrates that mrc is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost drosophila proteins with c h zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw , pita and zipic) were found to interact with cp and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw , pita, zipic and cg . first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw , pita, zipic and cg revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project № - - ). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t and t concentrations in adult male wistar rats. fifty rats aged between and weeks ( ae g) were divided into five groups (n = in each group), and drugs were administered each day intraperitoneally (ip) for days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol ( . mg/kg, n = and mg/kg, n = ) and clozapine ( . mg/kg, n = and mg/kg, n = ). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture hours after the last injection. the t and t plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v . . kruskal-wallis and bonferroni tests were used. t plasma concentration levels significantly differ between sham (median= . mg/kg) and haloperidol ( mg/kg) (median= . mg/kg), haloperidol ( . mg/kg) (me-dian= . mg/kg) and clozapine ( mg/kg) (median= . mg/ kg), haloperidol ( mg/kg) (median= . mg/kg) and clozapine ( mg/kg) (median= . mg/kg) groups (p < . ). however, no significant differences between the groups regarding to t plasma levels were observed. in conclusion, haloperidol and clozapine increased the t plasma concentrations, but didn't have any significant effect on t plasma concentrations. p- . . - isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a . lm filter and incubated on lb agar medium for hours. based on differencies of colony morphologies, isolates were selected and purified for identification. s lipase activity assay was carried out by rhodamine b. all of the strains exhibited lipase activity. for determining the substrat specificity of isolates, different substrates were used; nitrophenyl-butyrate, -nitrophenyl-caprylate, -nitrophenyl-laurate, -nitrophenyl-myristate, -nitrophenyl-palmitate. results were measured spectrophotometrically at nm. all of the strains hydrolyzed -nitrophenyl-butirat, while there was no activity with -nitrophenyl-palmitate. bacillus sp. l was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l will be cloned and expressed for more analyses and industrial applications. p- . . - some quantitative aspects of hair follicle layers differentiation e. vsevolodov , , v. golichenkov , a. mussayeva , , i. latypov llc "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with mcm hair diameter for those with the hair diameter mcm the ratio of hair medulla diameter to hair diameter increases from . to . . the ratio of hair diameter to the diameter of inner root sheath increased from . to . . it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes % of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from to u/g pulp basis and the consistecy of the pulp was between and %. enzyme at the desired concentration was added to the pulp and the mixture was incubated at °c for hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as % pulp consistecy, u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p- . . - biochemical characterization of lipase from bacillus subtilis strain a from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec . . . ) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a is partially purified and characterized. bacillus subtilis strain a is isolated from olive factory from soke (aydin, turkey) and identified with s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with % olive oil (vol/vol) for hour at °c. after incubation, cells were harvested by centrifugation at , rpm for minutes, resuspended in mm tris-hcl (ph . ) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at °c. the enzyme was active and stable in the broad ph range of . - . and temperature range of - °c. bacillus subtilis strain a have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p- . . - investigation of pin as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a (pin ) as a potential nfib interactor. pin is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin implicating this domain in nfi-pin interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin proteins in hek t cells showed that pin precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin . we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. - - . p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during - period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy , open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were to years old and their average age was . ae . . ofthem ( . %) were under years of age whereas of them ( . %) were above years of age. number of pregnancies were scaling between to with an average of . ae . . of mothers ( . %) were not undergone amniocentesis, whereas babies with chromosomal abnormalities were detected among mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. ( . %) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with . , . and % concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses ( . , . % and %) in both species with percentages %, % and % in r. ridibunda and %, % and % in b. viridis, respectively, while in the control group it was about %. furthermore, delayed larval development was detected. thus, the larvae from the control and . % oil group reached gosner stage (gs) , tadpoles from . % and % groups were at gs- and gs- , respectively, by the th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p- . . - effect of catechin loaded plga nanoparticles on glioma cell line histone h t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h subtype and hyperacetylation of histone h tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p- . . - serum dickkopf- levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf- (dkk- ) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk- levels in obese and non-obese children and adolescents. materials and methods: the study included obese children and adolescents ( males and females) aged from to years and healthy normal-weight controls ( males and females) aged from to years. serum dkk- levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = . ). however, there was no significant difference between dkk- levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk- levels were not changed in obese children and adolescents. p- . . - transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib À/À precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia À/À hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the kb human cdo promoter region identified five nfi binding sites: one cluster in the first kb region, another in the . kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to % in hek t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd /ankk gene variants in eos development. in this study, eos patients and healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd /ankk taq a (rs ) and bdnf val met (rs ) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd /ankk rs polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = . , or = . ; % ci: . - . ). however, no significant relationship was observed in the genotype frequencies of bdnf val met polymorphism between eos patients and controls (p = . ). these results indicate that, drd /ankk rs genotypes may affect eos development. however, bdnf val met polymorphism may not be associated with eos. lack of association of bdnf val met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black ) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black ) was determined between and nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black ) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm , prm and their relationship with defective spermatogenesis. materials and methods: this study has been performed on infertile and fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm and prm . results: distinct levels of spermatozoal prm and prm mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm was reduced in (% ), and the mrna levels of prm was reduced in (% ) out of infertile patients.in the current study,we found statistical significant association between the prm expression and infertility (p < . ).although prm gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > . ). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm and prm themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf , myogenin) in white muscle were studied in salmon parr of age groups +, +, + in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr ( +) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings ( +). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age +. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age + and + then in +, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to +. there was no differences in expression level of myod, myf and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. - - . p- . . - lmp and lmp gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu , f. ozbas gerceker sanko university, gaziantep, turkey, gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide (lmp ) and low molecular weight polypeptide (lmp ) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome . these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp and lmp gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp and lmp genes were determined. results: a deviation from the hardy-weinberg equilibrium (v = . ,p < . ) was found for the genotype distribution of lmp gene polymorphism, while the lmp genotypes found to be distributed (v = . ,p > . ). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp and lmp genes should be increased to understand their effects as a genetic marker. p- . . - investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have - nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found . ; . ; . nm respectively. the morphological features were examined by sem images. antioxidant activities of q , q ve q nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: - - -gep ". p- . . the effect of environmental enrichment on spatial memory and certain nmdars, and ht a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and ht a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into groups as enviromental enrichment (ee, n = ) and standard cage control (scc,n = ) groups. eeg housed in an enriched environment and sccg were kept in standard cages for weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr a, nr b, ht a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the training days showed that eeg located the hidden platform significantly earlier than sccg on days , (p = . , p < . ). also eeg significantly spent lower time in the outer zone of the maze on days , which was the sign of low anxiety level (p = . , p = . ). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < . ), in fst comparison of groups showed no difference (p > . ). the levels of nr b and ht a were significantly increased as compared to sccg as well (p < . , p = . ). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr b and ht a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p- . . - effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, ( weeks old) were divided into groups as control (cg, n = ; + , male+female) , experiment (msg-low dose, e g, n = ; + , male+female) and experiment (msg-high dose, e g, n = ; + ) groups. msg was administered at mg/kg/d to e g, . g/kg/d to e g for weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au autoanalyser. level of total protein was significantly increased in e g and e g groups when compared to cg (p < . ). level of alb€ umine was also increased in both egs but significant difference was seen in e g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < . ). although the glucose levels in both egs were increased, the increase in e g was statistically significant (p < . ). the alt levels of in egs were also increased but the significant increase was seen in e g (p < . ). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. ( mg/kg) + tartrazine ( mg/kg) + brilliant blue fcf ( mg/kg) + ponceau r ( mg/kg) + azorubine ( mg/kg) + indigotine ( mg/kg) + erythrosine ( mg/kg). artificial food color mixture were administered to g and g and drinking water was applied simultaneously to g by oral gavage per day for weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > . ). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g were higher than g and g in serum and kidney and brain homogenate (p < . ). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p- . . - er quality control protein network in cf to modulate f del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo and gst-pi enzymes. for this purpose, mrna and protein expression of nqo and gst-pi enzymes were analyzed in ht and sw (colon); hepg and huh (liver); pnt a and pc (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo protein was expressed in all cell lines and relative protein expression is highest in the hepg ( %) and pnt a ( %) while huh ( . %) showed relatively low expression of nqo . in addition, nqo mrna expression was relatively high in ht ( . fold) and pnt a ( . fold) when compared with liver cell line hepg ( . fold). gst-pi protein expression was found very high in huh ( %) while there was no expression in hepg . gst-pi mrna expression was relatively higher in pnt a ( . fold) and ht ( . fold) when compared with huh ( . fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo and gst-pi are ht and huh , respectively. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p- . . - tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about out of every cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. mrna studies and mirna study, which were designed for different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ . , fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf transcription factor and hsa-mir- . in the literature hsa-mir- and klf are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb ) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd , cd , cd and cd as positive markers and cd as a negative marker. gene expression profilling was investigated using affymetrix hg-u -plus arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < . , fold change≥ ). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of genes were identified that differentially expressed in msc line versus primer mscs ( upregulated and down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] association between loss of q , gain of q . and progression in sporadic colorectal cancer n. belder , b. savas , m. a. kuzu , i. pak , h. s€ umer c ß elebi , a. ensari , h. € ozdag biotechnology institute, ankara university, ankara, turkey, school of medicine, ankara university, ankara, turkey, ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix . genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from stage i, stage ii and stage iii samples. paired cnv analyses were performed using partek genomic suite . and genomic segmentation algorithm was performed using a minimum of markers per segment, a signal-to-noise ratio of . and the cut-off value for the gain and loss was set of ae . . the adjusted p-value ≤ . were considered to be significant. whole genome cnv analysis revealed that amplification of q . with genes was found the most frequent ( . %) in stage ii tumors. the most frequent ( %) amplifications were q . and p . in stage iii tumors. while deletion of chromosome q . in stage iii with a frequency of % was found the most frequent loss, deletion of q . was seen the most frequent ( . %) in stage ii tumors. two tumor suppressor genes smad and smad which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of q . might have a significant role in the progression of cancer. loss of q comprising two tumor suppressor genes is also another important finding. q loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no: s . p- . . - meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin , cin and cin according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin , cin and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin , cin and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin , cin and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with common genes showed that these targets were significantly enriched (p < . ) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group ), dia-betic+glibenclamide (group ), diabetic+plant extract (group ), untreated control (group ) and control+plant extract (group ) groups (n = for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract ( mg), and the reference drug glibenclamide ( mg/kg) by gastric gavage every day for weeks. in group , blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group , serum glucose, alt, ast, triglyseride and hba c levels decreased compared to group while cholesterol and ldl levels were high. in group , serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba c were higher compared to group . body weights increased except group and hdl levels were not altered. histologically degenerative changes observed on pancreas of group were decreased in groups and . there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p- . . - on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p- . . - qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan , c. neuschl , r. alberts , g. a. brockmann school of medicine, istanbul kemerburgaz university, istanbul, turkey, department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) , , , and . most likely candidate genes within or near the regions with the highest significance levels were identified. the genes f rik, gbe , a n , and four genes cenpc , stap , uba , gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr for fat mass, on chr for fat mass gain and on chr for lean weight, and chr for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to healthy women aged - years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than . . . % of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = . ) and protein (p = . ) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = . ), isoleucine (p = . ), leucine (p = . ), lysine (p = . ), methionine (p = . ), cysteine (p = . ), tryptophan (p = . ), and glutamic acid (p = . ) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = . ). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at nm, but in contrast to pvx, did not have additional peak at nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant - - ). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k , k , k and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a- dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full -utr and -utr sequences of m satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p- . . - physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga ) and with encapsulated antigen (plga ) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga particles (plga - . %, plga - . %). the flow cytometry results demonstrated that % of plga particles conjugated with egfp-sbb, and only . % of plga particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga particles size was . nm and plga - . nm. nta data also showed that mean plga particles size a little smaller than plga ( . nm and . nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p- . . - a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt , a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at °c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt cells with the isogenic cho-k cells as a control. cells after transient transfection were incubated at °c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt and the wild type control cell lines are performed to validate the reported model system. p- . . - host-pathogen interactions: is there a relationship between tlr polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than million individuals die from tb and each year , cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher ( . / , ) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even % of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study tb patients and healthy controls were included. tlr genotyping for rs , rs was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be . . results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p- . . - lytic action of bacteriophages as a tool for the obtaining of images p. boltovets , r. radutny , t. shevchenko institute of semiconductor physics nas of ukraine, kyiv, ukraine, scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the . % agar with the nutrient medium and the . % agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t were applied. samples were incubated during the twenty-four hours at + °c. after that stencils were removed and the samples were stained by coomassie blue r- or fuchsine (with further fixation by the % acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type (st ) complexes with ctx-m gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli - / -g and attc e. coli-reference strain and (ii) to compare the proteomes of strain at two conditions: with and without antibiotics. strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein , cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p- . . - molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu , i. gheorghe , , m. popa , , a. ion , m. mihai , v. lazar , , m. c. chifiriuc , carol davila" university of medicine and pharmacy, bucharest, romania, research institute of the university of bucharest-icub, bucharest, romania, faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of s. aureus clinical strains isolated in from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of . %, meca in . %, sscmec type ivd element in . %, ccrb in . % and sccmec types i, iii, iv in . % of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa ( . % of the isolates), followed by clfb ( . %), fib ( . %), hlg ( . %) and bbp ( . %). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p- . . - analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin ( %), vancomycin ( %) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than antibiotics. it was determined that among the investigated strains % contained meca, % -mecc, % contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in . % of cases, sed - %, seg - %, sei - %. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in % of strains, beta peptide toxin genes in %, delta toxin gene in %. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p- . . - impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] host-specificity determinants of bacteriophage vb_ecom_fv considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of water buffalo milk and dairy products ( water buffalo cream and water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso - and isolates were confirmed for the presence of the s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in of water buffalo milk ( %), of water buffalo cream ( %), and of water buffalo cheese ( %). a total of isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of isolates, ( %) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in out of ( %) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea ( / ; . %), two isolates produced sec ( / ; . %), one isolate produced ( / ; . %) sed, one isolate produced ( / ; . %) see and three isolates produced sec+sed ( / ; %) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet - . . ) and this article was part of a phd thesis. p- . . - identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il- receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp ; was cloned in fusion with an n terminal cleavable his-tag. the recombinant protein, his- was purified using nickel affinity chromatography. was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of was investigated by in vitro pull-down and size-exclusion chromatography. , a amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant his- was produced with a yield of mg/l culture. a structurally stable kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that also form dimers, supporting that it is a btp. current research is focused on solving the structure of and investigating its interaction with myd . might play a direct role in reduced immune response against h.pylori by binding to myd analogous to other btps. further characterization of will provide the first solid evidence of presence of a tir domain protein in h.pylori. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il production by bone marrow-derived macrophages k. korneev , , e. sviriaeva , lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor (tlr ) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il by bone marrow-derived macrophages from c bl/ mice, using tlr -deficient macrophages to control for specificity of tlr signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk / activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p- . . - apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz , l. monturiol-gross , m. j. pineda padilla , c. araya-castillo , a. alape-gir on bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc ( - ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with . lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of . triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor (her ) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for % of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb (mdr- ) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb- was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with , and nm of the drug respectively along with lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr- gene expression with the treatment of dox. especially after nm of dox treatment, mdr- was overexpressed to be fold when compared to control. in conclusion, it was demonstrated that mda-mb- cells have shown to display elevated resistance to higher doses of dox. p- . . - targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa , t. kondo toyama prefectural university, imizu-shi, japan, university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from °c to °c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-( -aminopurin- -yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached - %). nucleosides, such as ribosides, -deoxyribosides, and arabinosides were obtained in high yields ( - %). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el- (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant - - ). p- . . - dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim , n. demir , m. yildiz health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s -). also, cytotoxicity of the compounds against hepg cell lines was assayed by the mtt ( -( , -dimethylthyazolyl- )- , -diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s . also, schiff base complex had cytotoxic activity towards hepg cell line, that it was found to be more potent than the control cisplatin. p- . . - single particle electron tomography of rnap elongation complex, stalled at position + genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering or bp into the nucleosome; ec+ or ec+ , respectively). hear we produced two complexes, both stalled in the + position, one without break in the dna, and the other with introduced ssb at position + of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae °in a jeol microscope at kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+ complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant # - - . colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to who report new incidence rate of crc in turkey is . % among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh À g>a (rs ) promotor substitution, mlh g>c (rs ) exonic substitution, mthfr c t (rs ) and apc t>a (rs ) were investigated in this study. these snps "rs , rs , rs , rs " are located on p . , q , p respectively. colonoscopic investigations were performed on both cancer and control group. the snps were genotyped using kompetitive allele specific pcr technology in cases and healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these of the snps in mlh , mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh À aa genotype in group aged ≥ was found to be . % in cancer versus % in control group. moreover apc a, mlh c alleles were detected only and allele respectively. previously, apc a allele was determined in . % of a turkish cohort. however in the present study apc a allele was detected on allele only. studies showed mlh À promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c t and mlh g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p- . . - excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil and neil , have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either -oxoguanine ( -oxog) or , -dihydrouracil (dhu) by e. coli fpg and nei and human neil and neil from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases ( to ), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil and neil efficiently excised dhu located inside a bubble. fpg and neil was generally more active than neil in excision of -oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil also have shown activity in d-loops with rna. the presence of an additional unpaired -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf ( - - ). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas ( bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+ ), fab-dc/nflu(À )) or unrepairable (nflu/nflu (+ ), nflu/nflu(À ), nant/nflu(+ ), nant/nflu(À )) structures. besides, it has been shown that model dnas bearing bulky lesions in opposite positions (fab-dc/nflu( ), nflu/nflu( )) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(À ), nflu/nflu (+ ), and nant/nflu(+ )), whereas the level of specific excision from nflu/nflu(À ), nflu/nflu(À ) and nant/nflu(À ), nant/nflu(À ) was approximately % of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex ( bp) with fab-dc has decreased structurally dependent affinity for xpc-hr b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+ ), fab-dc/nflu (À ), fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(- )) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized - [ -( morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. - [ -( -morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p- . . - target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil and neil , and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg + . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil and neil showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf ( - - ). p- . . - does causes mhz magnetic field application kras and p mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group. mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for weeks as minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < . ).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for weeks minute/day exposure to mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p mutations in colon tissue. p- . . - synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g motifs) with the potential to form a spatial structure, gquadruplexes is known. g motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed samples of pancreatic ductal adenocarcinoma and samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. - - of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from healthy controls and patients with mm. the mean age for controls and patients were . ae . and . ae . years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a lung adenocarcinoma cells. a lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for a cell line. compounds , and which were including -chlorophenyl, -nitrophenyl on pyridine ring; -fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic values lower than . ae . lg/ml. compound showed the highest cytotoxic activity with a ic value of . ae . lg/ml, whereas cisplatin ic values were . ae . lg/ml lg/ml against a cells. cytotoxic activity of compound and with a ic value were . ae . and . ae . lg/ml, respectively. also, compound showed the highest population of early apoptotic cells ( . %) of the tested compounds which was . -fold higher than for cisplatin. compound produced a comparable population of apoptotic cells with a percentage of . %, respectively according to cisplatin's percentage of . %. it was determined that synthesized compounds , and had considerable anticancer activity against a cell lines compared to cisplatin. compound including -florophenyl on pyrimidine ring was the most cytotoxic compound against the a cell line. our study results demonstrated that compound , also induced apopototic pathway on a cells. p- . . in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl- cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ , , -trimethoxyphenyl = -methoxyphenyl > dillapiol > -methoxyphenyl> , dimethoxyphenyl > , , , -tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic values ranging from . (a cells) to . lm (mda-mb- cells). compounds, containing , , -trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to lm. anti-proliferative effects of isoflavones observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones demonstrated cytotoxicity in human pbmcs, up to lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing , , -trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag- is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl- , hsp /hsc family, growth hormone receptors, raf- , ubiquitin machinery and dna to regulate cell survival. for this reason, bag- is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag- are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag- silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag- enhanced cisplatin or paclitaxel-induced apoptosis in mcf- cells by down-regulating antiapoptotic and upregulating proapoptotic bcl- family proteins, changes on cell cycle, upregulation on subg phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag- has a suppressive role in pi k and akt signaling pathway in mcf- breast cancer cells through inhibition of akt phosphorylation and downregulation on pi k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag- silencing. bag- silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc and rapamycin-sensitive mtorc complexes, respectively. knockdown strategies of bag- is important to enlighten the network interactions of bag- and clarify its interaction partners in the cells. therefore utilization of bag- targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p- . . - biological activity evaluation of new , , trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately % of all female and % of all male cancer deaths in . the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. -[ -( , -bis( -substituted phenyl)- , , -triazin- -yl)piperazin- -yl]- -[benzimidazole/benzoxazole/benzothiazole- -yl)thio]ethanone ( - ) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and -chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt ( -( , -dimethylthiazol- -yl )- , -diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic values between - lg/ml. the early and late apoptotic cell percentage was determined as . for compound by flow cytometric analysis. the lox inhibition activity was found . ae . for compound . compound bearing -chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic / , ic and ic x . p- . . - single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal -drp group in oligonucleotide dna substrates in the presence or absence for me + ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants - - -a and - - -mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u mg) were seeded into the well plates and cultured with dmem supplemented with % fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in / (v/v) dilution ratio was given to first group, lm etoposide was given to second group, / (v/v) diluted rosemary and lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly % , etoposide reduced the viability by nearly % and rosemary with the etoposide reduced the viability by nearly % . the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing -( -aryl- -oxoethyl)- -substituted benzimidazole moiety on c glioma. c glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard ( -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for c cell line. compounds , , , , and , which were including hydrogen, -methyl, -methoxy, -chloro and -floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic values lower than lg/ml. compound showed the highest cytotoxic activity with a ic value of lg/ml, whereas cisplatin ic values were lg/ml against c cells. cytotoxic activity of compound , , , and with a ic value were , , and lg/ml, respectively. compound , and showed the highest population of early apoptotic cells as . , . , and . % respectively compared to cisplatin ( . %). also, compounds caused dna synthesis inhibition depend on their ic values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c cell lines. however, compound , and including -methyl, -chloro and -floro substituents were the most active compounds against the c cell line. also our study results showed that compound , , induced apoptosis in c glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc- and du prostate cancer cells were treated with rutin ( - um) and/or trail ( ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc- and du- prostate cancer cells were treated with rutin for and hours incubation and ic doses for hours incubation were determined um and um respectively. treatment with rutin, pc- cells is more sensitive than du cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc- and du prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc- and du prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p- . . - determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst- ) methods; and antigenotoxic effects against ccl on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration ( and lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations ( , , . lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst- , ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k and aml cell line hl- were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl , bax and caspase ), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p ) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k cell line but not in hl- cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k and hl- cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl and increasing bax and caspase levels. these effects of bor were observed to correlated with increasing the p expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p- . . - analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with - kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome p . , p . , q , q , p . , q . , q . , q . , q . , p , q . , q . , q . , q . , q . , q . , and q . , and also common gains were detected in chromosome p . , q , q . , q . and xq . in colon cancer dna samples, common losses were detected in chromosome p . , q , p . , p . , q . , q . , q . , q . , p . , p . , q . , and q . , and also common gains were detected.in chromosome q . , xp . , xp . , xp . , xp . and xq . both in gastric and colon cancer dna samples, common losses were detected in chromosome q . , q . , and p . , and common gains were detected in xq . discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase (parp ) recruitment which is a sensor for single strand breaks in dna. parp catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for to seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes and . bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt and a benzoxazole derivative xt b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k s) and imatinib-resistant (k r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl- , bad, bim, bcl-xl and mcl by real-time quantitative pcr. results: treatment of k cells with xt increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt b was not found to have similar effects on k r and k s cells. combined application of xt increased cell death in the mtt assay. mtt assay demonstrated that ic for xt treated cells in k r with imatinib (ic = . ) is lower than k r without imatinib (ic = . ). discussion and conclusion: our results showed that combining xt with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh and msh . alteration in expression or a defect in mlh or msh can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k s (sensitive) and k r (resistance) were grown in rpmi- . k r cells were maintained in rpmi- medium supplemented with lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh in k r cells is dramatically lower than equal amount of imatinib treated k s cells, whereas msh expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh genes leads to imatinib resistance. p- . . - characterization of interaction between rad inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france -diisothiocyanostilbene- , -disulfonic acid (dids) has been largely used during the last years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad -mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad recombinase protein. p- . . - mechanism of tap beta-mdm autoregulation p is a transcription factor which is the member of a p family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p , p is rarely mutated in tumors and elevated p expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm is known as the key player in regulation stability and activity of p . in addition, p induces mdm transcriptional activity, and caspase- , activations which cleave mdm n-terminal at asp . cleaved form of mdm binds p and promotes its stabilization. mdm suggested as a candidate to modulate p activity and stability too. however, an interaction between p and mdm has not defined well. in this study, we aimed to analyze the role of mdm in p stability. to define this relationship, firstly, we overexpressed the tap beta isoform using trex system in hep b. tap beta and mdm protein levels were determined by western blot. to examine whether mdm mediate tap beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg for hours prior to analysis. previous studies showed that p -induced caspase- and caspase- activation cleaves mdm . considering this, we firstly examined caspase- activation by western blot in hep b tap beta cells. then we analyzed expression of cleaved mdm and tap beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap beta-induced full-length mdm- expression. furthermore, tap beta enhanced cleavage of mdm via increased caspase- activation. in addition, inhibition of caspase- activation caused a decrease in cleaved-mdm levels in parallel with tap beta expression repression. our results suggested positive regulation between mdm -tap beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p is a transcription factor which is the member of p family. it has two promoters; while p promoter expresses apoptotic ta isoforms, p promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p . it has been shown that both tap and dnp isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap and dnp affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap isoforms via western blot in all hcc cell lines. then we overexpressed the tap beta isoform using trex system in hep b and snu cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; . only tap alpha isoform is expressed in a few hcc cell lines. . there is no correlation between basal expression of p isoforms and drug responses in hcc cell lines. . there is no change in expression of p isoforms after treatment of drugs. . we showed that the ectopic expression of tap beta in hep b arrested the cell cycle in g / s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p- . . - biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease (ape ). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape and archaeal mth revealed conserved amino acid residues in the nir-specific ap endonucleases ape , mth and exoa that are absent in xth. based on these data, we constructed four ape point mutants y h, n q, g s and t d and examined their dna substrate specificities. results obtained from biochemical characterization of ape mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll ( - °) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between and °. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p- . . - ercc /xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group (ercc ) is one of the important dna repair genes.ercc codon and polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated ml blood for biomarker testing. ercc genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v . ). there was significant difference between the frequencies of ercc polymorphism in cancer cases and controls (p < . ). the frequencies of ercc gln allele were . % in controls and . % in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or= . , %=ci . À . ). the above findings indicate that the genetic polymorphism in the ercc codon is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv .x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv . -kcsa-kv . chimeras was developed by us. this approach rested on high-level expression of kcsa-kv .x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv . -channel blockers that combines bl (de ) cells expressing kcsa-kv . and rhodamine-labelled agitoxin (rh-agtx ) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv . was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv . . for system validation, wellknown kv channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx , osk , and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv . channel binding using a number of agtx mutants constructed by us, whose affinities to kcsakv . were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv . channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv . -blockers for scientific and medical purposes. the work was supported by the grant - - from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp- ). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp- by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of : achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp- cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp- cells were was exhibited to possess the highest values of around % at ar concentration of lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis- inhibition of ire signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire (inositol requiring enzyme ), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u glioma cells. it was shown that inhibition of ire leads to up-regulation of the expression of krt , cd , mest, cenpu, myl , ing , ing , mybl , and mybl genes at the mrna level in u glioma cells, with more profound changes for mest, mybl , and cd genes. hypoxia leads to up-regulation of the expression of cd , ing , and ing genes and to down-regulationof krt gene in glioma cells. at the same time, inhibition of ire modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing gene, eliminates hypoxic regulation of krt , cd , and ing genes in glioma cells. the present study demonstrates that inhibition of ire enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase (pol ) of thermus scotoductus k isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol gene of t. scotoductus strain k recently isolated from an armenian geothermal spring. the draft genome sequence of strain k was deposited under accession number ljjr . . genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~ . kb) were ligated into the pet- b(+) vector (novagen) and transformed into chemically competent top escherichia coli. inserts were sequenced with t prom and t term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol gene from k shares % and % similarity with pol of t. scotoductus sa- ( . kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide ( l- fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of l- fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf- breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf- human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst- method. the most effective extract in inhibition of mcf- cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic ( ) values of lg/ml and . lg/ml at h. and h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf- cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf- cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf- cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, . ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, -aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than %. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than %. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for hours daily for one ( ximo), two ( ximo) or ten ( ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a -adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc a and ppargc b, significantly decreased in adrenal cortex of ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp , the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc a, ppargc b, nrf and nrf a). nonselective b-adrsblocker attenuated this effect. the selective a -adrs antagonist did not change adrenaline-induced stimulation of ppargc a, ppargc b, nrf and nrf a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a -adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis- generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for hours daily for one ( ximo), two ( ximo) or ten ( ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (Δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between Δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in ximo group. the mitochondrial mass in leydig cells from ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst- assay was used to evaluate the effects of metformin and tq in dhl (b cell lymphoma) and k (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci< ), additive (ci= ) or antagonistic effects (ci> ). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl and k cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl and k cell lines, ci values were . and . , respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with -hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with -hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after weeks of adipogenic induction in the presence of the chromium complex with -hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with -hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh and gfh . while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g a (pt g a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total participants were included in this study, patients with dvt and healthy blood donors. in order to identify fvl, pt g a, mthfr c t and mthfr a c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in patients fvl was present in ( . %) patients while in controls fvl was present in only ( . %). frequency of fvl was significantly higher in cases as compared to controls (p < . ). pt g a mutation was present in patients ( . %) and in healthy participants ( . %). mthfr c t and mthfr a c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c t/a c was found in patients ( . %) and in healthy controls ( . %), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c t and mthfr a c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c t/a c with fvl may be associated with increased risk of dvt. p-mis- self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t , tx ) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. - - r_center_a ( no. - - r_center_a ( - no. - - r_center_a ( ) and - - mol_a_ved ( no. - - r_center_a ( - . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk / or btk- inhibitors. the phosphorylation of erk / and btk- was determined by western blot analysis. proteomic analysis was performed by ultimate rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef ( v/cm). comparative analysis of bc and lc cells proteomes revealed about differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk- is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk- phosphorylation in lc than bc cells. in contrast erk / activity was not necessary for electrotaxis of lc cells and ef did not induce erk / phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre / /b/nz / , poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce , n. y. saral , f. b. aksungar , , a. coskun , , m. serteser , , i. unsal , acibadem labmed, istanbul, turkey, acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between and were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be lu/ml for cut off point and lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of lu/ml in ( . %), between and lu/ml in ( . %) and above the level of lu/ml in ( . %) babies, respectively. recall rate was . %. ch cases of neonatal tsh levels greater than lu/ml were . the incidence of ch of this group was : . there were no significant differences in the number of congenital hypothyroidism between males and females (p > . ). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou , m. kazou , v. alexandraki , b. pot , e. tsakalidou agricultural university of athens, athens, greece, institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of , , bp with a g+c content of . %. the genome contains , protein-coding genes on the chromosome covering up to . % of the genome sequence, trna and rrna. according to the subsystem-based annotation, , protein-coding genes were assigned to metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = , . % of total protein-coding genes) and protein metabolism (n = , . % of total protein-coding genes). furthermore, three prophage regions were detected; one intact ( . kb), one incomplete ( . kb) and one questionable ( . kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type diabetes mellitus (t dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after month. also, a healthy control group (n = ) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = . ) between the irisin levels of the patients at the beginning and after month treatment. a similar result was found between the control and the treated groups (p = . ), while a significant difference (p = . ) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in % of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. - - of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant - - ). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn in ammonia-exposed mouse cortical astrocytes (bisi; lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn and sn , second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r a and orexin ox heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin a and b (ghs-r a , ghs-r b ) for ghrelin, and orexin and (ox , ox ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r a and ox receptors to form heterocomplexes. formation of ghs-r a -ox receptor heteromers in transfected hek t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r a and ox receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r a -ox receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = ). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than . nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than . . our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva , , b. bekmanov , , a. amirgalieva , k. dosybaev , , z. orazymbetova , , r. zhapbasov , a. zhomartov , n. zumadillaev , n. zumadillaev llp "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than . ) there were found rarely met fragments (frequency lower than . ). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) breeds ( populations) were characterized. informative value and resolving capacity of the sum of str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. informative markers were chosen for both two breeds, markers being used for both breeds, while other markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele of bm was met in birlik population of edilbay breed as often as in % of animals while in two other populations there were no this allele. in kumtekey population one can meet % animals having particular locus (dyms ), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of preeclamptic mothers and mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk ( vs u/ml, p = . ). while tos levels were significantly higher in the preeclampsia group ( . vs . lmol/l, p = . ), the tas levels were significantly higher in the control breast milks ( . vs . mmol/l, p = . ). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis- investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included subjects ( female and male with age range of - years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at nd, th and th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = . , p = . ), tg (r = . , p = . ), tc (r = . , p = . ), ldl-c (r = . , p = . ) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis- eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than , compounds, incl. confirmatory and counter screening, ic/ec determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in , but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate -hydroxy-nicotine ( hnic) produced by a. nicotinovorans pao was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate hnic by using a genetically engineered a. nicotinovorans strain. the growth and the hnic accumulation were compared for two strains: . a. nicotinovorans pao wild type strain and . a genetically engineered a. nicotinovorans pao strain (part ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of hnic were monitored by hplc using a mn nucleodur - c ec column and . m sulfuric acid at a flow rate of ml/minute. the growth curve of the part ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of hnic are observed. although the sds-page analysis of the total protein extracts from the part ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a fold accumulation of hnic in the growth medium. the first attempts to purify ndh from the part ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part ndh strain for hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeÀinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of . - lm, and ache in the range . - lm. five cinchonidines displayed a - times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda ( lm) for h increased the expression of kir . mrna and decreased that aqp -and gs mrna. the expression of kir . was decreased by h exposure to glu ( mm) and tnfa ( ng/ml). at h incubation, nmda induced a decrease of kir . expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr subunit, and decreased that of nr b mrna. glu decreased the expression of out of subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir . mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to pediatric nurses and adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x . the average age of participants was ae years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was % and after the training it was %, which showed an increase of %. the preanalytic error rates which were . % in february were decreased to . % in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was nm and had a sufficient stability over a period of months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g ) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g dna is considerably more susceptible to lightinduced modification than duplex dna; -terminal tetrads of the g dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. - - from the russian science foundation. resistin levels in denervated obese rats n. saglam , t. ahmedi rendi , c. kahraman , a. alver department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study experimental groups were formed each consisted of rats. during weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < . ). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est gene is regulated at the translation level by + programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf p (ampk) and gcn p (eif kinase) on the prf rate in est gene. prf rate of est gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf , or gcn genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn p, and snf p, respectively. prf rate of est gene was measured as % in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than -fold, giving less than % prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by -fold in the wild type cells and prf rate increased to %. when the prf rate was analyzed in gcn and snf mutants, frame shift rate of est was - % in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn p and gcn p, positive regulator of gcn p, is also essential for the regulation of prf in est in response to stress conditions. it is clear that the basal level expression of est is highly dependent on the gcn p kinase complex. gcn p is also associates with ribosomes, indicating that gcn p might have a significant function in connecting the stress signals to biosynthesis of the full length est peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf p and gcn p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = ; nonerosive-nerd, n = ; healthy controls-hc, n = ) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee ( . ae . ng/mg.prt) versus hc ( . ae . ng/mg.prt, p = . ). mpo tissue activities in ee group were significantly lower ( . ae . u/mg.prt) than hc ( . ae . u/mg.prt, p = . ) while mpo serum levels were higher in ee ( . ae . ul) versus hc ( . ae . ul, p = . ). tissue chit levels were three fold increased in ee versus hc (p = . ). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=À . , p = . ) in tissue. nfkb and ecad levels had a positive correlation in serum (r = . , p < . ). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from , patients (mean age ae sd, . ae . ) were used for the correlation (excel analyze-it v . . ) and linear regression (pasw statistics v . ) analysis between the measured parameters. there was a significant (p < . ) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp* . ) + ( . *age) + ( . *wbc) + ( . *neut%)À . . correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of . . our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c -dox ratio ( : and : ) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp ad molt- ) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h o -induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to , tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < . ) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < . ). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil ( -fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of -fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of -fu synthesized via "click reaction", namely f -[{ -( ″, ″, ″, ″-tetra-o-acetyl-b-dglycopyronosyl)- h- , , -triazole- -yl}methyl] -fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in ml of solvent for h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta , ta (for the detection of frameshift mutations) ta and ta (for detection of base pair substitutions) according to the oecd guideline . the drug was solubilized ( lg/ml) with no precipitation in lutrol Ò -f /ethanol/water ( . : . : . , wt/wt) micelles ( . ae . nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the healthy goats were divided into groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t? (r ), at t = . s (r ), t = s (r ) and t?∞ (r ). this work was supported by the russian scientific foundation ( - - ). all animals had - % fat content. the contents of total protein ( . %), albumin ( . %) and urea ( . %) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol ( . %) and creatinine ( . %) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r (r = À . ), r (r = À . ), r (r = À . ); total protein or albumin levels with r (r = À . ), r (r = À . ), r (r = À . ); aminotransferase activity with r (r = À . ), r (r = À . ). the correlations were found between the total protein and albumin levels with k (r = . ), k (r = . ); glucose levels and r (r = . ), r (r = . ). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of > were classified as the high gs group (hgg), and those with a gs less than were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of . lmol h o equiv./ l for serum tos levels predicted high gs with a sensitivity of % and a specificity of %. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was . (sensitivity % and specificity %). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci , c. velioglu , e. recber , c. unsal , g. gulsoy , b. sariyar akbulut marmara university, istanbul, turkey, istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis . the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis cells were subjected to lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for hours but then cells resumed their regular growth. upon addition of lg/ml (-)-roemerine to the bacillus subtilis cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono- , -lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf- breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf- breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase (hplscr ) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr . this study aims to identify the region of hplscr that interacts with topo iia. to identify the topo iia interacting sites in hplscr , nterminal deletion constructs of hplscr viz Δ -hplscr , Δ -hplscr , Δ -hplscr , Δ -hplscr and Δ -hplscr were generated by pcr, cloned, overexpressed and purified to homogeneity using ni + -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex p- and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr and the gst-ctd-topo iia. the binding region in hplscr will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr . Δ -hplscr did not show any enhancement of the decatenation activity compared to full length hplscr . hence, the binding region could be in the - region of hplscr . further deletions were done in the - region of hplscr as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr that binds to topo iia. we conclude that hplscr interacts with and enhances the activity of topo iia and the - region of hplscr is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr and the physiological relevance of this interaction in the cell. a. ugur kurtoglu , v. aslan , e. kurtoglu department of biochemistry, antalya education and research hospital, antalya, turkey, department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs . is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs . , ivs . , ivs . and ivs . . our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis- investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen , h. kara karadeniz technical university medical biochemistry department, trabzon, turkey, balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba ),non-lysosomal b-glucosidase (gba ), cytosolic b-glucosidase (gba ),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose b-ltyrosine- -naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba .inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba was purified at . -fold with . % yield.gba was illustrated and kda at sds-page.ic value of ampicillin/sulbactam antibiotic for gba was found . mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba was illustrated and kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba .ampicillin/sulbactam antibiotic inhibited to purfied gba at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu , p. b. sermikli , a. abbasi taghidizaj , e. yilmaz ankara university, institute of science, ankara, turkey, ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, t -l cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type- diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft , ft , vitamin b and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b , folate. the cases were - years old, male individuals who admitted to our hospital between april -march for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: ) were divided into four subgroups; group (g) : - . lg/dl, g : - . , g : - . , g : ≥ . from these, the number of cases whose th levels were measured (n: ) given respectively; g : , g : , g : , g : cases. also the number of cases whose vit b and folate levels were measured (n: ) given respectively; g : , g : , g : ,g : cases. levels of pb were determined by icp-ms. th, vit b , folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t , tsh and vitamin b (p > . ). on the other hand there was statistically significant difference between t and group , , (p < . ) but there was no difference between group (p > . ). the average folate belongs to the first group was about % higher than the other groups, and found that the difference was statistically significant (p < . ). there are many publications which have various results between pb levels and t ,t , tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b , folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b . but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged ( ). since that time this method became associated with a number of major breakthroughs in the field ( ) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors ( ) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf - - . ( ) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. , , À . ( ) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. , , - . ( ) katritch v., cherezov v., stevens r.c. ( ) . annu rev pharmacol toxicol , - . p-mis- stamp is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp knockdown. interestingly, in the crpc cell line rv , where-stamp knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of d thin layer chromatograms using d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional ( d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional ( d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for d plates, and cannot be adapted to quantitative evaluations of d plates. in this study, the applicability of the gel documentation systems and d analysis software for the analysis of d tlc plates were examined. material and method: d tlc of lipids: st dimension: methyl acetate/n-propanol/chloroform/methanol/ . % kcl ( / / / / v/v); nd dimension: chloroform/methanol/acetic acid/ water ( / / / v/v); detection: charring. d tlc of aminoacids: st dimension: . % (v/v) formic acid; nd dimension: toluene/glacial acetic acid ( : v/v); detection: uv. phospholipid and aminoacid standards, each include different classes were developed by d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest d gel analysis software. for the method validationa) plates containing same lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample b) plates containing same lipid classes were developed in different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates results: volume of each phospholipid and aminoacid had less than % intra and inter-assay cv. conclusion: gel documentation system with d gel analysis software can be used for the quantitative analysis of the d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, adult male wistar albino rats were used. rats were separated into four groups as control rats (g ), mm nac (g ), mm nac (g ) and mm nac (g ). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding mm kcl to the organ bath. then, in the first minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at . . results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < . ). similarly, nak atpase activity is also decreased in a dose dependent manner (p < . ). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk , which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of . - . . the optimum temperature for keratinase activity was investigated by varying the incubation temperature between °c and °c. optimum keratinolytic activity was observed at °c and ph . . the enzyme was stable at °c. the activity was investigated in the presence of some chemicals, including sds, tween , dmso, triton x- , edta, nacl, zncl , cacl , glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel ( %) using standard molecular weight marker and estimated about kda by sds-page. the keratinase isolated from bacillus uk could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of bthalassemia and a-(including two of . kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler Ò instrument. the a/b-globin mrna ratio of each sample was calculated based on the Àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis- self-assembling peptides mimic supramolecular biochirality r. garifullin , , m. o. guler bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis- pon activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon- activity in both total and hdl subgroups, and evaluation of performance characteristics of pon- activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed obese, overweight and normal subjects. fasting morning blood samples were taken from all study groups. hdl subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl -c concentrations were calculated with the subtraction of hdl -c from total hdl-c. hdl -c and total pon- activity were determined by using eckerson method. non-hdl pon- activitiy was calculated with subtraction of hdl pon- activity from total pon- activity. results: total hdl-c, hdl -c and hdl -c concentrations and the activity of total pon- and hdl pon- were found lower in obesity according to overweight and normal subjects (p < . ). negative correlations were found between body mass index and hdl -c, total pon- and hdl pon- (r = À . , p < . ; r = À . , p < . ; r = À . , p < . , respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl -c measurements, pon activity, measurement of total and hdl -c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis- hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > . was accepted as significant value. results: in a study of blood donors ( male, female), the overall prevelance of anti-hev igg antibodies were . %. the blood samples were collected different areas. the prevelance of anti-hev igm antibodies was . % and he was years and acting a butcher during years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were %, the animal husbandry were % and the veterians and the butcher were % were found. discussion: the prevelance of anti-hev in the north cyprus ( %) was found low such as the prevelance of the turkey ( %). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january and january . clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that blood samples were sent to our laboratory in one-year period. . % of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause ( %). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be %, %, %, %, % and % respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of patients ( female, male) aging . ae . years ( - ) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of g sugar ( hour), -hour and -hour. these parameters were statistically analyzed in comparison to simultaneous hba c results. glucose measurements at hour had better distinction power (p < . ) between these individual groups than initial and -hour glucose measurements. the average hba c (%) levels for healthy, pre-diabetic and diabetic individuals were . ae . , . ae . and . ae . , respectively. roc curve analysis showed . % sensitivity and . % specificity for the clinically accepted hba c cut-off value of . %. hba c cut-off value of . % had a higher sensitivity of . % and comparable specificity of . %. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at -hour after sugar administration in ogtt test as opposed -hours generally used for diagnosis. low sensitivity was observed for the clinically adapted . % cut-off value of hba c. the cut-off value of . % for hba c was found to be more sensitive with comparable specificity than the . % cut-off values for diabetes screening in our population. our results suggest that . % for hba c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin ( mg/kg, po) every hours for a total of doses prior to an apap challenge ( mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp e , cyp a , or cyp a in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf -dependent pathway. we found that stamp fused to cyan fluorescent protein (cfp-stamp ) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp with various fluorescent protein markers revealed that cfp-stamp puncta corresponded to lipid rafts that were labelled with caveolin- -rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp and caveolin- -rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp internalization. notably, stamp was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c ), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with -hydroxy- -nitrobenzyl bromide (hnbb) that covalently modifies the trp residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity ( %) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states -phosphofructo- -kinase/fructose- , -bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose- -phosphate (f p) to fructose- , -bisphosphate (f , bp) and vice versa, and f , bp is an allosteric activator of phosphofructokinase- (pfk ), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb - ), pfkfb is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb , variant- and variant- , coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g d)induced oncogenic transformation on pfkfb expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb mrna and protein expressions (mrna; ipde: ae . ; ipde+kras: . ae . and protein; ipde: ipde+kras: . ). we then, checked individual expressions of splice variants and observed that while pfkfb splice variant- (p -v ) expression was reduced by k-ras induction (ipde: ; ipde+kras: . ), pfkfb splice variant- (p -v ) expression was increased (ipde: ; ipde+kras: . ). then, we checked effects of p -v and p -v on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb variants increased f , bp concentration (p -v : . ; p -v : . fold; compared to empty vec), glucose uptake (p -v : %; p -v : %) and glycolysis (p -v : %; p -v : %) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb isoforms and observed that both pfkfb isozymes localize to the nucleus, with more prominent nuclear localization of p -v compared to p -v . also, nuclear localization ratio of p -v increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p map kinase inhibitors on mda-mb- cell line introduction: p mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p mapk sb and sb on mda-mb- cells to further elucidate the controversial role of p mapk on cell proliferation or cell migration. materials and methods: mda-mb- cancer line was cultured in rpmi- supplemented with % fbs. the cytotoxic and cell migration effects of sb and sb inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb- cells were determined by icelligence system. results: it was found that sb p map kinase inhibitor was more effective than sb . however, no significant effects of low doses of lm and lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to hours as determined by icelligence system. on the other hand, both sb and sb significantly prevented cell proliferation at a concentration of lm. both sb and sb significantly reduced cell migration in a time-dependent manner at a concentration of lm. then, we tested whether each p mapk inhibitors have any effect on cell adhesion during a treatment period of hours using icelligence system. only lm concentration of sb reduced cell adhesion for about . hour (p < . ). conclusion: p mapk inhibitors sb and sb differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no - . mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto , f. zilifdar , s. yilmaz , t. sarac ßbasi , i. yalc ßin , n. diril hacettepe university, ankara, turkey, ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of , -benzoxazine- -one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta and ta strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s ) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a d-common features pharmacophore model with hiphop method. according to the results, only bs , bs , bs and bs exhibited strong mutagenic effects on both strains in the presence and absence of s . additionally bs , bs , bs and bs (in the absence of the s ), bs , bs and bs (in the presence of the s ) showed weak mutagenic effects on ta . hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r and hydrophobic groups at the position of r of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since .the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = ) were evaluated in dried blood samples which sent to our laboratory for the study between august and august . electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms ,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in ( . %) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in ,organic acidemia in ,fatty acid oxidation defects in patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain- are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr /nr b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr /nr b subunits, calpain- , hsp and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain- are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp that reduces the affinity for ca + of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain- . our data suggest a physiological role of calpain- at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca + influx, the activated protease regulated by hsp , promotes the removal of nmdar from the plasma membranes, decreasing ca + entrance through this receptor-channel and protecting cells from ca + overloading. tissue transglutaminase (tg ) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg mutant constructs. renca cells were transduced by lentiviral particles encoding wttg , transaminase-defective tg -c s form with low gtpbinding affinity, gtp-binding deficient form tg -r a, and transaminase-inactive tg -w a. in order to investigate the role of tg transamidating and gtpase activity in cell migration, scattering assay was used where colonies for each mutant clone was followed for a time interval of hours. our results showed that non-transduced control and tg -c s mutant renca cells demonstrated a similar migration pattern with a % of scatter activity. on the other hand, % colonies formed by renca cells overexpressing wttg and tg -w a mutant scattered away from each other. a small insignificant increase in scattering was seen in % of the total number of colonies for renca cells overexpressing tg-r a construct. data from this study supports that gtp-binding activity of tg is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = ), subclinical hypothyroid (n = ), subclinical hyperthyroid (n = ). serum tsh, free t (ft ) and free t (ft ) levels were determined by chemiluminescence immunoassay and serum -hydroxy (oh) vitamin d (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, . to . miu/l), ft (range, . to . ng/dl) and ft (range, . to . ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t and t levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t and free t levels. results: subclinical hyperthyroid group had significantly higher (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < . ). (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co , no x and so x (ghorbani et al., ) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., ) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr , ts and ts were used as ethanologenic microorganisms. expression of vhb in ts is lower than in ts strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts strain by up to . x cfu per ml of fructose ( %, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s ( ) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology : - . ghorbani, f., younesi, h., sari, a. e., najafpour, g. ( ) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. ) . in this work, e. coli strains fbr , ts (low level vhb expressing) and ts (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for hours at °c and rpm in a ml erlenmayer flask. the ethanol production was improved over % by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as . , . and . for fbr , ts and ts strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. ( ) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., ; geckil et al., ] . in this study, ethanologic e. coli strains fbr , its derivative strains ts (vgb+) and ts (vgb+) were used. ts was constructed in such that it could express more vhb than ts . bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations ( . - mm) were used as inhibitors from lignocellulose hydrolysate. . mm acetic acid was used as an inhibitor. the growth of vhb expressing ts and ts strains was inhibited about % after hours fermentation time. strain fbr was inhibited as high as % by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd , an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd on adipogenesis in t -l preadipocytes. notably, azd treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of t -l preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription- (stat- ) during adipocyte differentiation. furthermore, azd largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd inhibits adipogenesis in t -l preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat- . effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = ) and food colouring (fcg, n = ) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than . were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment ( . ae . ) and control ( . ae . ) groups showed no statistical difference (p = . ). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr c ) polymorphisms and unipolar or bipolar depression. the nr c gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr c (bcl and rs ) in recurrent depressive disorder (rdd) patients. methods: our study included volunteers with recurrent depressive disorder and healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr c gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs ) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl- (g/c) were as follows: cc % and %, gc % and %, gg % and %, respectively. there was not a significant difference when the frequency of the allele (p = . ) and genotype frequency (p = . ) were compared between the patients and the control. genotype distribution in the rs region (a/t) of the patients and controls were tt % and %, ta % and %, aa % and %, respectively. allele frequency (p = . ) and the genotype frequencies (p = . ) were not significantly different among the groups. conclusion: numerous nr c gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr c polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy y human neuroblastoma cell line. sh-sy y cell line treated with - lm tq by solving medium for , and h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm , p , p , akt, pten, cdk , cyclin d , caspase- , - , - , - , bcl- , bax, parp, bcl-xl, bid, dr , dr , puma, noxa, mmp- , - , timp- , - and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt profiles array data analysis by using student's t test. ic value of tq in sh-sy y cells was detected as lm at th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm , akt, cdk , cyclin d , bcl- and mmp- . it is also observed that tq caused a significant increase in the expression of p , pten, caspase- , - , bid, dr , puma, noxa and timp- . it was also found that tq in sh-sy y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. - - . the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm wild type (wt) and its isogenic Δmacab mutant strains. we also used h o survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab- xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h o . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after hours of growth in lb-broth there is at least -fold increase in expression in response to the presence of h o . on the protein level macab can be detected starting from hours of growth in lb-broth and it reaches maximum expression on hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h o properties. antiproliferative and apoptotic effects of noscapine on mcf- and mda-mb- human breast cancer cell lines approximately - % of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor . these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf- and mda-mb- human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf- and mda-mb- cell lines were analyzed by roche xcelligence system. the cells were cultured in % fetal bovine serum containing dulbecco's modified eagle medium at °c in a humidified atmosphere containing % co . h after seeding, the cells were treated with different doses of docetaxel ( . to nm), tamoxifen ( . to lm), and noscapine ( . to lm). cultured cells were harvested, fixed with % formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at lm thickness and stained with h&e, ki- , bcl- , cyclin-d , and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf- cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf- cells, hovewer docetaxel and noscapine were more effective on mda-mb- cells. tamoxifen has more apoptotic and antiproliferative effects on mcf- cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb- cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a -ml centrifuge tube by a neodymium magnet through a -cm cushion of % sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was -fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. - - . epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl sandwich elisa and rt-pcr. the mechanism of action of zeb / on ccl promoter has been studied by luciferace assay and chip. ccl coding region was cloned into pcdna . and stably transfected into dld- cells. ccl deficient ct cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl and sip expression was studied using ihc or flow cytometry. emt status catagorised crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl in induced dld-sip cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl in the induced dld-sip cells as well as mesenchymal crc cells as compared to epithelial ones (p = . ). promoter studies showed that zeb / bind to ccl promoter and and activate ccl gene expression. no metastasis was observed for dld- cells overexpressing ccl but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl is up-regulated by emt inducing transcription factor sip , and mesenchymal (metastatic) crc cells secrete significantly more ccl compared to epithelial (non-metastatic) ones. ccl did not induce emt per se but abundant secretion of ccl by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for - minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of . to . lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the lg/plate concentration s. typhimurium ta . at all the doses antimutagenic response was significant at (p < . ) against both the strains with a percent mutagenicity decrease from to for ta followed by ta with percent antimutagenicity from to . the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [( , , , )-tetrathia[ . ] ( , )- , , -thiadiazolophane; ( , )dioxo- , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane; ( , , , )-tetraoxo- ( , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane and ( , , , , , )-hexaoxo- ( , , , )-tetrathia [ . ] ( , )- , , -thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for hours. at the end of the each hour, cell vitality was assessed by measuring the absorbance ( nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of : of . mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using , -diphenyl- -picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than ng/ml or greater than ng/ml were accepted as inappropriate initial testing. fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of patients who referred to the hospital phlebotomy unit were collected using holder into the different tubes. first tube was ml glass tube and with no additive, second was ml tube with gel separator, third was ml tube with gel separator. serum was separated and immediadiately analysed for biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft , ft ) which are the most common requested laboratory tests. thyroid function tests of patients are analyzed in ankara numune education and research hospital in october -may . five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below % of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for -month study period was . %. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis- development of polyclonal and monoclonal antibodies against fatty acid binding protein (fabp /ap ) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp ) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp protein made by recombinant dna technology. recombinant his-fabp was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp , as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb- , mcf- and cd + /cd -cells and ic values of thz were found to be lm, . lm and lm respectively, at hours. invasion potency of mcf- , cd + /cd -and mda-mb- cells were determined as %, %, . % when compared to relevant treatment controls. migration potency of mcf- , cd + /cd -and mda-mb- cells was determined as . %, . %, % respectively. among the three cell lines mda-mb- cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin- , b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb- , mcf- and cd + /cd cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. healthy - year children's ( girls, boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = . , p = . ) and a negative correlation with humidity (r= À . , p = . ). mean urine density in spring was higher than that of autumn (p = . ) and winter (p = . ). mean value of summer was higher than autumn (p = . ) and winter (p = . ). - months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis- implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of pharmacies located in north cyprus were involved in the study with %stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two ( . %),responded positively to this demand. it has also been identified in the study that . % of the daily sale of antibiotics in the first half of the year was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with . % and upper respiratory tract with . %. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be ( . %),compared with the studies conducted in greece,italy,malta and spain % and egypt . %that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic ) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a cells including with ultrastructural and morphological effects. ic value of aa on a cells for h was detected with mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a cells in dose-dependent manner and ic on a cells was found to be . mm for hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on immunoassays studied by the beckman coulter unicell dxi immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of tests were measured by the beckman coulter unicell dxi . d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin ( . , . , . , . , . and . g/l hemoglobin) and intralipid ( . , . , . , and g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid ( %, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au . after spiking the pools, the tests were measured again in duplicate on beckman-coulter dxi analyzer. a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b , testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca . , ca , ca . , insulin, pth and e , and of lipemia for progesterone, ca . , vitamin b and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb andiso plus tak- . treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase- (pon- ) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p mapk and nuclear factor erythroid -related factor (nrf ) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p mapk, phosphorylated c-jun n-terminal protein kinase (pjnk / ) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk / ) and nrf expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb treatment. p mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p mitogen-activated protein kinases, sb , tak- . silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were males in each groups which contained patients with silicosis (group ), individuals having similar symptoms with silicosis from same occupational area (group ) and healthy subjects (group ). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev , fev %, fev /fvc, fev / fvc%, fef - and fef - %) were compared. the average age of group , and are . ae . , . ae and ae . years, respectively. there was a significant difference between group and in terms of the ada level (p < . ). there was a negative correlation between ada activity and fev , fev %, fev /fvc, fev /fvc%, fef - values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. - - . the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar , m. tekin , s. civelek firat university, elazig, turkey, cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by genus on the world and by genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega . program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, ll of internal standard (d - deoxycortizol) in methanol was added to ll standart or serum and centrifuged at . rpm for minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at • c and dissolved in mobile phase. ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. k ) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value , , intercept value À . and r² value . . the regressione quation was elisa= À . + . lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste polycomb repressive complex expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u , h. s€ urer , g. bilgin , d. y€ ucel ankara training and research hospital, medical biochemistry department, ankara, turkey, ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. patients with copd, patients with asthma and healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha- antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < . ). there was no significant difference for ferritine and alpha- antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova , , v. timofeev , , n. zhukhlistova , y. abramchik , t. muravieva , r. esipov shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, national research centre "kurchatov institute", moscow, russia, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at k from single crystal grown in space (sp. group p ) was collected on the spring- synchrotron-radiation facility (japan) and the structure was solved at . a resolution, using the molecular replacement method (pdb id i c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id da ) and human pnp (pdb id pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h ) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h . ara h aptamer was used to recognize any residual peanut major allergen protein ara h in food samples. recombinant ara h protein was produced and puirifed to be used as a target. ara h aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between and . kg/m . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of women. students with high bmi (> ) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (< ) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (< ) student group. waist circumference, percentage of body fat was higher in high bmi (> ) group than low bmi (< ) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (> ) and bmi (< )). students were grouped depend on their waist circumference. healty individuals who had lower than cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum ( , , aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions ( % ethanol and polyethylene glycol -peg %) and different method (ultrasonic treatment w/ o c/ minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at o c/ hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %), followed by liquid samples treated by ultrasonic method prior to transformation ( . %). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: patients, having delivery beyond weeks, and who did not have any other medical problems were included in the study. the patients had normal g. oral glucose tolerance test results. they were divided as groups, based on their newborns' birthweight as " g. and g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low ( . ng/l, . ng/ml and . ng/ml respectively), normal ( . ng/l, ng/ml, . ng/l respectively) and high ( ng/l, ng/ml, g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin ( . , . , . , , . and . g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access analyzer. the hemolysis indices were measured on beckman coulter au . a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations ( . ng/ml), and a negative interference for myoglobin at low concentrations ( . ng/ml) and high concentrations ( ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥ . g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a- , glycosidase enzyme with an incidence of : , - : , . as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a- , -glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a- , glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of patients's examined, of them were diagnosed with pompe disease depending on clinical findings & low a- , glycosidase activity. enzyme activities of pompe patients were . nmol/ml/hour as lsd suspected patients'activities had a mean of . nmol/ml/hour (p = . ).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher ( vs - u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group ( vs u/l, p = . ). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = . r=À . ) was observed between a- , -glycosidase and ck activities (n: ). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of ( h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of ( h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f ) on biofilms formed by s. aureus atcc and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic ( mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of mg/l of f the mic of kanamycin was decreased -fold, and the mics of both erythromycin and ampicillin were lowered -fold. at the concentration of mg/l f also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto and propidium iodide. the addition of f to preformed h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f at concentration of mg/l led to death of up to % of biofilm-embedded cells. this fact suggests that f efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project № - - and the german academic exchange service (№ ). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged and over is - times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin- secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed young and elderly patients with pneumonia and their respective aged matched controls (n = , n = ) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml and . ae . mg/l, . ae . ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < . and p < . ). elderly patients with pneumonia have significantly higher crp levels than their controls (p < . ). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin ( . g/dl) and normal calcium ( . mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of ml were aliquoted. purified albumin, . g, was added to one of these pools and albumin concentration was determined as . g/dl. the low and high albumin pools were mixed at different ratios and pools with . , . , and . g/dl albumin concentrations. total calcium and albumin concentrations of these pools were measured at a beckman-coulter au analyzer and ionized calcium was measured at a radiometer abl blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of . g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: . %, . %, . %, and . %, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: . %, . %, . %, and . %, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at > g/dl albumin concentrations. ionized calcium is significantly affected by . g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba c and fasting glucose concentration was investigated. lp(a), hba c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july to july . there were patients with these test results at the same time. the patients were grouped according to hba c values: group i < . % (n = ), group ii . - . % (n = ), and group iii > . % (n = ). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba c (r = . ; p < . ), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < . . there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < . ). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep -p is written in kit inserts. target values ( %) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from to mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate . % from the target when the degrees of hb are mg/dl. when the degree of hemoglobin is mg/dl, the test strays about . %. potassium levels increase ( %) at mg/dl hb while this increase reaches to . % at mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases %, at the mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. d-structure of the hu protein from mycoplasma gallisepticum have been determined with a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund ( - - ). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox . sox is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with -ethyl- -( -dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range ( . - pg/ml) with a detection limit as low as . ng/ml sox . furthermore, the developed sox immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox antigen and anti-sox antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: z ). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: . women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh ( - ) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= . -( . age)À( . fsh) + ( . lh)À( . estradiol) is detected and the model's r = . is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld- ) and endometrium (ecc- ) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic ) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at and lg/ml concentrations by hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld- and ecc- . however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca - against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca - is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+ %psa. tissues were seeded on mm petri dishes containing dmem/high glucose supplemented with % (v/v) fcs and incubated %co in air at % relative humidity and at °c. after seeding, the medium was unchanged for days and then it was changed in every days for days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over %) in culture when compared with the negative control ( %). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g /g phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g /g phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g /g phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, g ). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe o ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used - -trimethyl- -phenyl , , hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between and °c simple nanoparticles decreased the membrane fluidity. at physiological temperatures ( - °c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at °c, and at °c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. -isoprostane is considered as an indicator of oxidative stress. mao activity and -isoprostane levels were measured in some diseases. however, there are no information on -isoprostane levels and mao activity in newly diagnosed patients with stage hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and -isoprostane in newly diagnosed patients with stage hypertension. the study included newly diagnosed stage hypertensive patients with no other systemic disease. patients were selected as randomized ( women, men; range of age - years) and healthy individuals as control ( women, men; range of age - years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and -isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > . ). compared to the control group, the activity of mao and -isoprostane levels were found significantly higher in patients (p < . ). conclusion: increased -isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, -isoprostane p-mis- determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c -a guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from , males, , females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma and sigma subunits did not affect rna elongation. we showed that the sigma -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp /dis , dis l and dis l proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the s subunit at °c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the s ribosomal subunit, concomitantly with the increase of s and s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of -thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of -thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip- / modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov- g and tov- d. for this purpose, tov- g and tov- d cells were treated with limnio grape skin extracts at a range of concentrations, at °c, for , and hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov- g and tov- d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl , bclx, bax, bak and bcl l , along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. % of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse , gse and gse . we analyzed two datasets of er(+) breast cancer patient samples (n = , n = ) compared to normal breast tissue (n = ) using apadetect and sam. a total of utr shortening and utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb- cells that are otherwise of triple negative nature. our results suggested that most of the genes are utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between - bp also detecting the impurities in dna solution in %, % and % ratios to the dna of interest. the developed method was validated using the in-house dna fragments of , and bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, . lm, . mm id . cm and tskgel dna-npr, . lm, . mm id . cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for bp in the range of . ng to ng dna with the regression coefficient of r = . . lod and loq for the bp dna was found to be . ng and . ng, respectively. the recovery values for the %, % and % impurity ratios were found . . . and . , respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = . ). the length distribution of small rnas revealed nt-peak corresponding to trna-derived fragments, amount of which was increased by . -fold under dtt treatment. the trna isotypes that gave rise to almost % and % of all nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of nt fragments produced from these trnas are precisely phased halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor (rnh ). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun , , , j. kim , , j. y. hong dankook university, cheonan, south korea, institute of tissue regeneration engineering (itren), cheonan, south korea, the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; hour, day, week, month and months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage ( hour to week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at hour or day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p- . . - utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco- cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco- cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day ) caco- cells and tested sample apa events by rt-qpcr. utr isoforms were identified by using race pcr. we identified genes ( % of all apa events) to undergo utr shortening in differentiated cells compared to proliferating cells. on the contrary genes ( % of all apa events) went through utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine- of rna polymerase ii c-terminal domain by end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y -s -p -t -s -p -s at the c-terminal domain (ctd) of its largest subunit, rpb . phosphorylation of serines (s , s , and s ) and tyrosine- orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine- site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine- phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine- ctd phosphomarks using integrative structural biology. one of them, a factor involved in the -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine- that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t g human gbm cell line were used to evaluate the effects of ind ( lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p < . ), cell viability (p < . ), cell rate at s phase (p < . ) and g + m phase (p < . ), camp levels (p < . ), the levels of pdgfr-a (p < . ), mrp- (p < . ), nf-jb (p < . ) and cox- (p < . ) in comparison to control group. ind mildly increased apoptosis (p < . ) and caspase- levels (p < . ). interestingly, ind increased htert levels ( %, p < . ; %, p < . ). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox- and other tested factors. p- . . - interaction between fibrinogen and insulin-like growth factor binding protein- under physiologic conditions and influence of diabetes mellitus type on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp- , is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp- . besides glucose regulation, igfbp- stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp- , their change in dm type (dm ) patients and involvement in fibrin clot. samples from adult healthy persons and dm patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp- /fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp- . in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp- /fibrinogen complexes can be differentiated from igfbp- oligomers and igfbp- /alpha- macroglobulin complexes. the amount of igfbp- /fibrinogen complexes was lower in patients with dm . complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp- monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm was additionally able to bind exogenous igfbp- . since igfbp- stimulates wound healing, directly and by delivering igfs, igfbp- /fibrinogen complexes may be seen as igfbp- storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k q (q substitution for k in position ) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated (single mutant) and (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk- cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst- reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk- cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p- . . - his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova , m. tileva , k. maskos , i. ivanov , g. nacheva institute of molecular biology "roumen tsanev", sofia, bulgaria, proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five Ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p- . . - development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected ( - in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec . . . ) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta- , -d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall -fold purification, a . % yield and specific activity uÁmg À protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled kda. it was found that the enzyme displays pi . , prefers ortho-nitrophenyl-beta-galactoside as substrate (km mm) and shows maximum activity at °c and at ph . - . . the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km . . it was established that the gene carries an open reading frame consisting of bp ( amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family (cazy database). p- . . - different splice-forms of tdrd protein mutated in cataract's and glaucoma's interacts with s k / o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s kinases (s k) are important players in cellular pi k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s k -tdrd (trap). tdrd is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s k-tdrd interaplay and study its role in cells. bioinformatical analysis of tdrd sequence revealed the presence of potential phosphorylation sites of s k . using in vitro kinase assay, we have demonstrated that recombinant s k phosphorylate from fragments of tdrd . formation of s k -tdrd complexes in vivo was further confirmed by coimmunoprecipitation using anti-s k and anti-tdrd antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd co-localize with s k in hepg cells, predominantly in perinuclear region, enreached for one of the tdrd isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s k with methylated arg interfere with tdrd from hepg lysates. the physiological characteristics of s k -tdrd interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~ kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x- in high concentrations, but efficiently dissociates in the presence of m urea and mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of to kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a and other proteins. serum albumin, transferrin and protein with molecular weight , kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b fusion protein. we have shown that cytochrome b enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at nm in oxidized environment. cytochrome b change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b fusion tags multiples recombinant antibody yield (from to times) and doesn't affect antigen-binding properties. the bb - site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i- c (monab i- c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i- c in bb - of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb - , bb - and both their scrambled version, and bb - peptide. monab i- c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i- c was investigated by spr method using plasmon- device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb - , bb - , bb - , and the scrambled versions of bb - and bb - peptides on a binding to monab i- c and on the fibrin polymerization process. in spr analysis was showed that bb - and bb - peptides, but not their scrambled version, binds to monab i- c, immobilized to a chip. turbidity data showed that only bb - and bb - peptides caused the -fold decrease of the rate of the lateral association of protofibryls at the concentration . À m and . À m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb - site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp- ) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is - ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= , female= ) was . years (sd . years). among them ( %) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while ( %) as a hf-ref (hf with reduced ejection fraction). mean bnp values was . pg/ml (sd . pg/ml) and mean lvef was . % (sd . %). mean urine ngal was . ng/ml (sd . ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = . , p = . , wilcoxon signed rank test z = À . p < . ). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p subunit of splicing factor sf ;(iii) voltage dependent anionic channel- ;(iv) dynamin- ; (v) heterogeneous nuclear ribonucleoprotein a /b . obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near uridine derivatives were determined. the combined kinetic (nnna, , ) and structural data (acta crystallogr., d , , ) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as - kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp (forkhead box p ) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp on tumor cell migration. our results showed that foxp has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase (pde ) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde was increased in cell lines cancers thus pde inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde gene expression and cgmp signaling in the mcf- er + and mda-mb- er À . methods and materials: the ed of the hawe on both cell lines were examined by using mtt viability test then the expression of pde and cgmp concentration were measured in timedependent manner (in the ed ) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, lg/ml is ed for both cell lines and the hawe lead to reduction in pde mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf- and mda-mb- , cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having -stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to da in neutral ph but the pore is closed by going through a conformational change under ph . . as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg- s is obtained by adding amino acids to the primary chain in order to have a -stranded beta barrel porin. ompg- sl is formed by further adding amino acids to loop l and by replacing lysines with arginines. ompg- s and ompg- sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd . / . and infrared spectra are recorded. further, temperature profiling are recorded for the range between to °c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ °c, although this is less than the wt transition at $ °c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase (parp ) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt . further characterization of nudt will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group ( mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid ( mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups . % peanut oil was added throughout the experiment. the all rats were sacrificed at the end of weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in . % of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp- levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if , if and if . the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim p possesses a function of mtif . however, the mitochondrial translation has not been stopped in the yeast strain without aim p which is surprising taking into account the fact that if is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim p is almost indispensable for cox p synthesis, and that it affects the translation of cox mrna through its -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim gene than in the wild-type. taken together, our results point on the multiple role of aim p in mitochondrial translation: in addition to its function as mitochondrial if , it specifically regulates the amount of complex iv subunits and its activity. p- . . - the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of patients with pcos and healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < . ). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < . ). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). we found that bche hydrolyses this molecule at ph rather better than at ph . . at ph values, purified human bche has a km value that was times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately % that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p- . . - determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi auto analyzer. the epo levels of samples was . ae . mul/ml (ranged between . and . ) mul/ml. when we performed ae sd for the studies population we determined normal serum epo levels were as . - . mul/ml. the upper limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml and the lower limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included patients diagnosed with subclinical hypothyroidism and healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at À •c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were ng/l, control group ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > . ). apelin levels didn't show significant correlation with bmi (p > . ). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv /mia import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv is a sulfhydryl oxidase that partners with the import receptor mia to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv mutant strain (erv - ) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv s role in these processes. we discovered that the erv - strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv and mia mutants tested were similar to the wt control. in addition, while all the erv and mia mutants tested exhibit temperature-dependent defects in mia oxidation, only the erv - strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv - strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh ) that compromises gsh protein folding and/or stability. to address whether gsh deficiency in the erv - mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv and mia mutant strains. only the erv - strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv - are due to gsh depletion and neither erv nor mia play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are . - . ng/ml and - ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak Ò hplc and zivak Ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast l aaa column ( mm i.d). the mobile phase was composed of, a: mm ammonium formate (af) in water, b: mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c column ( mm i.d., mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p- . . - heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp expression and induced caspase-dependent apoptosis in c rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at °c for h in c cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase- activation in c glioma cells. pretreatment of the cells with non-toxic dose ( lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase- activation in c glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp , matrix metalloproteases ; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from mhz to mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain - amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp- and hnp- encoded by defa , defa and defa genes are firstly expressed in neutrophils. human hdp and hdp encoded by defa and defa are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd strain was cloned, expressed, characterized in escherichia coli blr(de ) strain, and then val- and phe- amino acids, supposed to be responsible for substrate preference, were substituted for met- and ile- amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe ile enzyme are %, %, %, %, % and % of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val met mrbsh enzyme are %, %, %, %, % and % of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d , t , and d ) but according to alignment is closer to chr even the last one motif is e , t , n . it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf - - . evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un , s. sahin , f. duygu university of gaziosmanpasa, department of biochemistry, tokat, turkey, oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (% - ). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha (ta ), thymuline and thymosin beta (tb ) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < . ) and plt and wbc levels were significantly lower in the cchf group (p < . ). levels of tf, ta and tb were found to be significantly higher in cchf (p < . ). there was no mortality during the study period. duration of hospitalization was . ae . days. levels of tb were significantly correlated with duration of hospitalization (r= À . , p = . ). alt levels were significantly correlated with tf levels (r = . , p = . ). patients received ffp and apheresis for the supportive treatment, while patients received only ffp and patients got only apheresis. patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > . ). we report survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic values were determined as and mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh of carbamate group of methocarbamol to the carboxyl group of asp side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser and ser residues of the pseudoenzyme. p- . . - dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc ) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than mm. moreover, primary cell cycle analysis has indicated arrestment at g /m phase in hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g /g and g / m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor alpha (hif a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor . it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif -a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of - % oxygen and - % nitrogen for week. rats in adm groups were injected intraperitoneally in a dose of . nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from - animals and adm, hif -a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif -a level. adm levels and hif -a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif -a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif -a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif -a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p- . . - covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, ( ) their circulatory-lives and stability (in different ph and temperature values) enhance ( ) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. postmortem skeletal muscle were analyzed hour periods in first -hour after death. nd and rd -hour periods were statistically significant (p < . ). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of , were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr b and akr b in hct- colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr b ; aldo-keto reductase family , member b , and akr b ; aldo-keto reductase family , member b . both are -kda cytosolic reductases that are similar in both amino acid sequence identity ( %) and tertiary structure with the (a/b) barrel topology. while hct- , a colorectal cancer cell line, cells expresses akr b robustly, there is no expression of akr b . in this study, we have stably knocked down akr b through shrna technology and overexpressed akr b in hct- cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr b , we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr b overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr b and akr b have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m medium (ph ) boosted nisin production at °c, as the addition of . lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p- . . - coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between and , the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to , , and amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf isoform in hct- colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf . we show that vegf undergoes n-linked glycosylation in hct- cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p- . . - novel methods for modulating the activity of bcl family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl- family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl- family, including the pro-survival members bcl- , bcl xl and mcl , and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl- family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl- family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl- family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora , v. castro-fern andez , c. a. ramirez-sarmiento , e. a. komives , v. guixe facultad de ciencias, universidad de chile, santiago, chile, department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt . elucidating the domain swapping mechanism of the forkhead domain of human foxp fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp follows a threestate n « i « u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp , showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt , ). p- . . - the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of . % per week for up to weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the -week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full -week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < . ). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = . , p = . , p = . , respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p- . . - pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck kinases on ents activity. methods: adenosine uptake ( lm adenosine, s, °c) was assayed in hk cells preincubated with mm or mm d-glucose for h and exposed to lm -br camp, lmh or lm tbb for h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent and ent contents were quantified by western blot. results: high d-glucose in hk cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h ) through an increased ent uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent or ent under normal d-glucose ( mm) or high d-glucose conditions ( mm). also, we saw that tbb (ck inhibitor) decreased the activity of ent and ent under normal glucose conditions, decreasing the localization of ent at cell surface, while the membrane localization of ent decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent . this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was . ; body mass index was . ; serum lipo (a) level was . and plasma fibrinogen level was . . there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = . ; p = . ), significant positive correlation between the serum lipo (a) and rdw-sd (r = . ; p = . ) and significant negative correlation between lipo (a) and mpv (r= À . ; p = . ). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is . kda as found. optimum temperature was °c and half-life times were , , and . min at , , and °c, respectively. optimum ph value was . . however, it shows significant ph stability at ph values . and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d lipase. therefore, the effect of immobilization on kinetic parameters of d lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c q tumor necrosis factor-related protein (ctrp- ), c q tumor necrosis factor-related protein (ctrp- ) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp- , ctrp- and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included patients with knee oa and healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp- , ctrp- and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp- in the patient group were significantly higher than in the control group (p = . ), serum ctrp- and kallistatin levels were not statistically different (in order of p = . , p = . ). in the patient group, there was not a significant difference between serum ctrp- , ctrp- and kallistatin levels and radiologic stages (respectively p = . , p = . , p = . ). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = . , r = . ; p = . , r = . ; p = . , r = . ; p = . , r = ). discussion and conclusion: serum ctrp- levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp- and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp- , ctrp- and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as - and - °c. in consequence of the salt test, the test isolates were grown at - % nacl. of thermophilic isolates were selected by rep-pcr and according to s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of - %. enzyme tests showed that, some of the isolates were able to produce protease (f , f , f , f , f and f ), amylase (f , f , f , f and f ), cellulase (f , f , f , f , f and f ), xylanase (f , f , f , f and f ) and lipase (f , f , f , f and f ). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p- . . - warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt a, du , hela, hep b, hek t, shsy y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter (glut ) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp- , - ) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco and casp- was higher in hek t compared to other cell lines. casp- was upregulated in cancer cell lines, mostly in hep b. glut- levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p has a repressive effect on glut , it also can lead us to study about p levels. the effect of inhibition of pi k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her , her ) play important roles in breast cancer pathogenesis. her /her interaction is the most potent heterodimer and it causes oncogenic pi k/akt activation. inhibitors of pi k/akt pathway (akt inhibitor and pi k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi k/akt inhibitor pi- on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb- cells (which has egfr overexpression and pten deficiency) and skbr- cells (which has her overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi k/akt inhibitor pi- for different time periods ( - h) . egfr, her her total rtks expression and pi k/akt pathway inhibition (p-akt and p-p s k expression) were evaluated by western blot. in mda-mb- cells, there were significant decrease in p-akt and p-p s k proteins' expression during the first h. this inhibition was followed by reactivation of the signaling pathway after h. in skbr- cells, p-akt and p-p s k proteins' expression were significantly decreased during the first h. the pi k/ akt signaling pathway in these cells were reactivated after h. basal expression of egfr and her in mda-mb- cells and basal expression of her and her in skbr- cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her and her expression levels. these results suggest that dual pi k/mtor inhibiton by pi- may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid -formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a cell line, respectively. for the whole cell transcriptome analysis a cells were treated with lg/ml of binase for h followed by mrna extraction and library preparation. sequencing was performed on solid xl wildfire next-generation sequencer. we found that binase internalized into a cells after h of incubation. the binase at lg/ml was absolutely non-cytotoxic towards a cell line and was active in the cell culture medium during h incubation. the analysis of rna-seq data showed that among thousands of protein coding transcripts transcripts were up and down regulated by binase, among them transcripts were induced and repressed. binase repressed the production of s a and tnxb which act cancer biomarkers, scn a and drd which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp , rasgrp and alpk transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. . ) is a specific group of phosphatases capable of hydrolyzing myo-inositol , , , , , -hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: -phytase, / -phytase, and -phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain . . showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. . . phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was °c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet a expression vector. multicopy plasmid pet a/agpp contained the sequence of the phytase gene of pantoea sp. . . under t promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag- (bcl- associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag- exists as three major isoforms (bag- s, bag- m and bag- l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp /hsc molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl- , raf- kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag- expression levels affect cell survival in mda-mb- (er, pr and her /neu negative) breast cancer cell lines. we first cloned bag- l gene to a cloning vector, later we transfected mda-mb- cells for overexpression of bag- . we also used bag- sirna to silence bag- gene. western blot analysis was applied to demonstrate relative expression levels of bag- , its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag- overexpressed cells to checkbag- 's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag- overexpression. in addition, our study revealed that once bag- forms a complex with c-raf/b-raf/hsp /akt/bcl- , modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag- and its isoforms are found, the role of each bag- isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type diabetes because of different physiological effects. in circulation, glp is cleaved by ddp in a very short time. if glp can be protected from cleaving, the effective time of glp would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp was performed by sds-page. the purity of fusion protein was found to be %, as confirmed by glp elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at °c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp . ) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology ( %) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with % homology was identified in the genome of clinical isolate of m. morganii . rt-pcr analysis showed that this gene had the maximum expression at h of growth. in addition, the cellular extract of m. morganii had small actinolytic activity. cloning of the gene into e. coli dh a cells led to the synthesis of the kda protein. it is known that the highest expression of serratia proteases is observed at h of growth, and the molecular weight of the mature proteins is kda. it was shown that metalloprotease gene of m. morganii expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight ( kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova , d. zhuravleva , a. laykov , k. forchhammer , a. kayumov kazan federal university, kazan, russia, eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only - % homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = lm) over atp (kd = lm) suggesting that they compete for binding to lbrglnk. neither -oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. - - a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k ) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k -fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c. . . . ) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in . u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a antibody, its kappa and lambda variants, in order to understand the difference on the active site between a and a which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a antibody kappa and lambda variants have been already determined, there was no structural information though about the a antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are s À and s À for a kappa and a lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi x . translation elongation factor ba (eef ba) is a component of a heavy form of translation elongation factor (eef h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor a (eef a) restoring its active conformation appropriate for aminoacylated trna binding. eef ba forms a tight complex with translation elongation factor bc (eef bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef ba. this suggests that the eef ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef a, eef a and eef a . we showed that recombinant eef ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef a comparing to full-length eef ba. similar effect on the catalytic activity of eef ba was observed after its interaction with eef bc. in contrast, the effect of full-length eef ba and its truncated forms on the rate of guanine nucleotide exchange in eef a was similar but relatively modest compared to eef a . this can be explained by higher rate of spontaneous gdp dissociation from eef a comparing to eef a and lower affinity of eef a to eef ba. thus, we propose that the n-terminal domain of eef ba via flexible linker region may interfere with eef a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef bc and eef ba n-terminal domains abolishes this inhibitory effect. p- . . - assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below %, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof + mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of proteins across all samples with variation across lc-ms replicates was % on average, and the second approach led to quantitation of proteins in total, of them across % of samples, all with the variation about % on average. p- . . - interaction of plasminogen fragments k - and k with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k - and k with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k - and k reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by - % in the presence of k - and k . fragments k - and k have high affinity to fibrin fragment dd (dissociation constant is . lm for k - and . lm for k ). analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k - interacts with complex of fragment dd-immobilized k as well as k with complex of fragment dd-immobilized k - . the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa - region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k - and k , which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p- . . - implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf ) cells expressing human dopamine d receptors using [ h]sch- and bodipy-fl-skf- as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf- itself were rapid with an apparent half-life of t / = . ae . s for association ( nm) and t / = . ae . s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas levels in gi cancer patients. female and male patients were included in the study (n = ): colorectal, gastric, pancreatic, liver, ampullary, gall bladder and esophageal. from all gi cancer patients, ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at g for min, and plasma samples were carefully removed and stored in À °c prior to use. the level of plasma gas was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas levels in cancer patients were determined as follows: . ae . ng/ml in colorectal; . ae . ng/ml in gastric; . ae . ng/ml in pancreatic; . ae . ng/ml in liver; . ae . ng/ml in ampullary; . ae . ng/ml in gall bladder and . ae . ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas levels. taken together, these results suggest that gas may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas would be an attractive therapeutic target for slow down the progression of gi cancer. monday september : - : computational biology p- . . - computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p- . . - computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g -dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g -dendrimer to mannose receptor (mr ) were performed using hex . . software. taa lumo atoms were conjugated to homo atoms of pg-g dendrimer using maestro software. results showed that pg-g -dendrimer displays carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g -conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g -dendrimer binds to the core of the mr receptor. increasing of the number of mannosamine conjugated to pg-g -dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, -deoxy-d-xylulose- phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the d structure of tpdxr by phyre . energy minimisation and root mean square deviation (rmsd) was performed by drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket . server a reliable d model for dxr from t. parva was modeled by using au as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be . and . a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p- . . - molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme (bace ) as a drug candidate for alzheimer's diseases g. yalcin , i. yildiz biotechnology institute, ankara university, ankara, turkey, department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme (bace ) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly , thr , gln , phe , tyr , lys , thr , arg , thr residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace . the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version . . . dst was determined by bpa - p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley . this work was supported by the russian scientific foundation (grant - - ). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r , r , r , r , k , k ): r = . + . * p À . * f r = . + . * p À . * f r = . + . * p À . * f r = . + . * p + . * f k = . + . * p + . * f k = . À . * p À . * f these formulas show that the maximum total effect of fat and protein contents influences at r and r . a significant coefficient (> ) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p- . . - molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv . channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv . with peptide blockers agitoxin and its mutant forms. d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code a h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv .x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. - - . simulations were performed using the supercomputing center of lomonosov moscow state university. p- . . - homology modeling and molecular docking study of the paraoxonase- and its polymorphic variants q/r and m/l for non-statin lipid lowering drugs paraxonase- (pon ) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon and its polymorphic structures pon q/r and m/l . fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon and its polymorphic variants pon q/r and m/l was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon (pdb code: sre). ns molecular dynamic simulation analysis was performed using gromacs . . . affinity of lipid lowering drugs to pon and its polymorphic variants was predicted by molecular docking approach using autodock . suite. unlike other lipid lowering drugs that they have negative Δg values for affinity, probucol, orlistat and betasterol was calculated by positive Δg values ( . , . and . kcal/mol). these values suggest that they may have no affinity to pon q/r polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon -m/l and sibutramine to pon q/r were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon and m/l polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon structures may reduce the activity of antioxidant pon enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over distinct functions in mammalians. the mammalian prlr gene consists of - exons and several and regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_ . , nc_ . and rn_celera, ac_ . ) were used and primary analysis showed that both sequences contain several gaps (varying from . to kbp), corresponding to about . % ( - kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_ . prlr gene has two exon- (one is about kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from . % to . % (from kbp to kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon- and - , similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the utr (exon- and - ). in rats, there are exon- variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share common non-protein coding exon- variants. in conclusion, it is found that the rnor_ . version of the prlr gene has the highest number of unidentified base pairs (corresponding to . % of the gene) and the second exon- is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase- enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon (pdb id: sre) from protein data bank. modeller software was used for homology modeling of pon and its polymorphic variants that's called as pon q/ r and m/l . amino acid sequence of human serum pon (uniprot: p ) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs . . software. molecular docking calculations on each of the polymorphic structure of the pon was performed with auto dock . . suite. for each substrate, y residue showed open conformation in pon and m/l polymorphic structures while q/r polymorphic structure showed closed conformation. in comparison between structures of pon variants, in most cases statins had lower affinity to q/r polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon . by considering that the high affinity drugs can have reducing effect of pon activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p- . . - self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar , d. alpay , a. r. atilgan , c. atilgan sabanci university, istanbul, turkey, northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever- m' and tensiometer 'bpa- p'. three groups of animals were formed from clinically healthy holstein cows at the age of - years according to the fat content in the milk sample. group i - cows (milk fat content . ae . %), group ii - cows (milk fat content . ae . %), group iii - cows (milk fat content . ae . %). this work was supported by the russian scientific foundation (grant - - ). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from . % to . %, lactose and mineral content varies from . % to . %, respectively. the dst parameters (r , r and k ) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r , r and k . obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p- . . - exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p- . . - effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas , , l. buimaga-iarinca , c. floare , l. janosi faculty of physics, babes-bolyai university, cluj-napoca, romania, national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl À pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~ % of all cancers and up to % in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c : -phosphatidyl-choline: di- : -phosphatidyl-choline: cholesterol : : ) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region - and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id n ) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant - - . serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a -ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at nm uv light. antibodies of rhd antigen were detected using differential pulse method at between . and . v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in rhdnegative pregnant women ( - th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon and of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p- . . - investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega and bioedit version . . . and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega and paup . b with bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui . using gtrgamma model with bootstrap replicates. beast v . . was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total reads (with an average read length of . bp). there were contigs having > bp sequence length whereas contigs with > bp sequence length. for further analysis we have taken contigs with > bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of À . ≥ % of total contigs were mapped to the reference with ≥ % contig match coverage. the community analysis revealed that domain bacteria is predominantly present ( . %) in the water sample, followed by eukaryota ( . %), viruses ( . %) and other sequences ( . %). most abundant phyla was proteobacteria ( . %) and the most dominant family was enterobacteriaceae ( %) followed by xanthomonadaceae ( %), vibrionaceae ( . %), pasteurellaceae ( . %), shewanellaceae ( . %). we performed functional analysis of all contigs using rapid annotation using subsystems technology (rast) which detected coding sequences and rnas in subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with % accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~ % accuracy by this single cleavage, with optimal combination of - cleavages improving the accuracy tõ %. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants - - , - - and - - ) and ras presidium program 'molecular and cellular biology'. p- . . - mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain - which was derived from soil-isolated b. pumilus p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to lg/ml) and ability to produce extracellular enzymes in quantities almost -fold higher than the parent strain. these features make the - strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus p and - strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. . % of total cdss differ in - strain in comparison to p one, % of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k n) leading to str resistance is identified in s ribosomal protein s p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain - gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided x ( p) and x ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) overall genome coverage. assembled genome sequences of p and - strains included and scaffolds > bp with a calculated genome size of bp and bp, respectively. the gc content was %. both draft genomes have been deposited at genbank (jojx . for p and jhud . for [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b and safr- ) revealed the max. ani value ( . % and . %, respectively). b. pumilus sh-b strain has been used as a reference for snp calling in strains p and - . snps for the p strain and for the - strain were classified as nonsynonymous variants. radical nucleotide substitutions from the - genome were not found in p genome. among them, the mutation in the codon of rpsl gene (coding s ribosomal protein s ) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a , that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a antibody is only m À min À , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a . the hybrid qm/mm method allows to study the reaction mechanism of interaction of a with paraoxon. it was shown that the reaction proceeds via the classical s n mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr- to paraoxon. qm/mm approach determines position for mutagenesis -leu- in light chain. for one of the mutant in this position -leu argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu arg exhibits a -fold increase in k /k d compared to a ( m À min À vs. . m À min À ). double mutant leu arg/ser ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu arg still binds paraoxon three times better, that may be due to the fact that the serine in position increase the nucleophilicity of tyr . thus, our results are in line with our computed predictions. this work was supported by rfmefi x . due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is x pixels and field of view (fov) is lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on % raw meat and % offal tissue. calculations proofed with imagejÒ. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday september : - : mechanisms of pro-inflammatory diseases p- . . - the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als mg/kg (oral) group, tor+als mg/kg (oral) group, tor/det+als mg/kg (oral) group, tor/det+als mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated °clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il- b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als and tor/det+als groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak project, coded s . ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type diabetes was induced in male c bl/j mice by i.p. injection of high-dose stz ( mg/kg b.w.). after weeks of stz-induced diabetes animals were treated with/without d ( iu/mouse per os) for weeks. blood serum ohd was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum ohd level, indicative of d deficiency. significant alterations in d -endocrine system were found as is evident from reduced expression of cyp a , cyp r , vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of ohd content and partial normalization of liver tissue structure were achieved after d treatment. d administration partially normalized expression of cytochromes involved in d metabolism and hepatic pro-inflammatory factors. d treatment prevented overexpression of rel-a and phosphorylated p /rel-a translocation to hepatocellular nuclei that is most likely mediated through , (oh) d and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d against diabetes-induced liver injury. p- . . - lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c c muscle cells s. savranoglu , h. ipek , s. arslan , h. delig€ oz , a. r. t€ ufekc ßi , i. demirtas , t. boyunegmez t€ umer graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c c myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt- (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine ( ) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c c cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser )/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the -deoxyglucose uptake assay. results: eae at and lg/ml significantly increased the glucose uptake and % compared to insulin resistant control cells. metformin at mm increased this parameter up to %. eae increased pakt ser /akt level - % and lpl expression - % for and lg/ml, in insulin resistant myotubes, respectively (p < . ). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt molecule through ser phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: t ) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, : n À ) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of . mg/kg ( mm/kg) daily during weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day . we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. - - ), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. - - mol_a. p- . . - circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd +/ cd + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd +/ cd + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd +/ cd + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥ . pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < . ) as well as high levels of aopps-alb, apoptotic emps (cd + /cd + /an-v + ), and nt-probnp (p = . , p = . , p = . respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd + /cd + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: patients who were diagnosed with obstructive sleep apnea and healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = . , p = . , respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = . ). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p- . . - decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type (dmt ) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt patients. the cross-sectional study included dmt patients, treated at the primary health care centre zenica from december to april , distributed into groups according to glycated hemoglobin (hba c) values: a (n = , hba c ≤ . %) and b (n = , hba c > . %). complete blood cell count, fasting blood glucose (fbg) and hba c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss . . p values fasting blood glucose and hba c were significantly higher in the group b compared to the group a (p < . ). there was no significant difference of nlr, plr and rpr between the groups (p = . ; p = . ; p = . , respectively). significant correlation of inflammation-based scores with fbg and hba c was found only between plr and hba c in the group a of dmt patients (r = . , p = . ). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type diabetes mellitus (t dm) and determine whether or not these are reliable markers for diagnosis. we evalueted people's results who were carried out oral glucose tolerance test (ogtt). acording to -h values of plasma glucose in the ogtt; . group (normal glucose tolerance: ngt): under mg/dl (n = ), . group (prediabetic: impared glucose tolerance (igt)): ranging from mg/dl to mg/dl (n = ), . group (firstly diagnosed diabetic by ogtt): above mg/dl (n = ). . group is clear diabetic without complication (taking treatment) group (n = ). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = . ) and plr (p = . ) values. nlr values are significantly higher in prediabetic ( . it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type diabetes mellitus (t d). this study aimed to analyze the long-term impact of altered metabolism in female t d patients at the level of mediators of inflammatory response. this study included femalet d patients and control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il- , were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = . ), crp (p = . ), il- (p = . ), leukocytes (p = . ) and sedimentation rate (p = . ) in female t d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba c (p = . ), il- and glucose ( . ), il- and bmi ( . ). in our study, female t d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il , crp and sedimentation. other studies conducted in female population associated elevated levels of il- and crp with t d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure , (oh) d levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included renal transplant recipients ( female, male; mean age: . ae . ) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month . serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, (oh)d and , (oh) d levels were measured. gfr values were estimated by ckd-epi. plasma , (oh) d levels were determined in a lcms- triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss . software was used for statistical analysis. results: although plasma , (oh) d levels significantly increased (p = . ), we did not find any significant differences for serum (oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = . , p = . for alp), we observed significantly higher calcium and gfr values (p = . ). vitamin d insufficiency was present . %, deficiency . %, severe deficiency % before transplantation, insufficiency was also seen . %, deficiency %, severe deficiency . % after transplantation at month . conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type diabetes (t d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type diabetic subjects. this study involved newly diagnosed type diabetics and healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. ra patients, as patients and healthy volunteers with mean age of - were included in the study. serum levels of vegf, angiopoetin- and tie- were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang- and no levels were significantly higher in the as group ml, p < . ; p < . ; p < . ). no differences were found between as and ra for tie- (p > . ). vegf, ang- , tie- and no levels were positively correlated in both as and ra patients (p < . ), but no correlation was detected between clinically activation index das- , basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > . ). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang- in as patients was sufficient (auc ( % cl): . , p < . ). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang- measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately - h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with patients and healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at g for min at °c. the serum samples were stored at À °c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p- . . - neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of patients with polycystic ovary syndrome and healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count ( . ae . vs. . ae . , p < . ) and platelet count ( . ae . vs. . ae . , p < . ) were higher in patients with pcos compared to the control group. lymphocyte count was . ae . in pcos patient and . ae . in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls ( . ae . , . ae . p < . , . ae . , . ae . p < . , respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b , caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b were measured in c bl mice with alf induced by single i.p. injection of aom ( mg/kg of b.w.) and after neutralization of tgf-b induced by single i.p. injection of ab-tgf-b ( mg/kg) h before aom injection. in alf mice, blood tgf-b was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s ) fraction by~ % and %, respectively, but was slightly depressed in the membrane (p ) fraction by~ % and~ %. aom induced an increase of postsynaptic proteins: psd- and nnos by~ % in p fraction. tgf-b neutralization resulted in a reduction in the expression of presynaptic proteins by~ % in s fraction and~ % in p fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd- and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b neutralization appear to be confined to the presynapse. % of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox- and cox- ) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox- and cox- , they have serious side effects on gastrointestinal system. therefore, new highly selective cox- inhibitors having fewer side effects are needed. in the present study, selective cox- inhibitory activities and cytotoxic effects of new series of -benzoxazolinone and thiazolo [ , -b ]- , , -triazole derivatives previously synthesized as specific cox- inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco- cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il- b, il- , il- and il- levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl- and tgf-b levels of patients with aa. patients with aa and healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl- and tgf-b levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b levels in the patient group were significantly lower compared to the control group whereas serum ykl- levels were significantly higher in patient group. tgf-b levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl- level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl- levels in aa patients suggests that ykl- plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin (il- ), and soluble intercellular adhesion molecule- (sicam- ) in comparison with control patients. cimt values are similar in psa ( , ae . *) and psc ( , ae . ) patients. however, both of them were significant increase compared with control ( . ae , ). regarding inflammatory markers il- serum levels in patients with aps was higher than pcs ( ae . ) and healthy controls ( , ae . ) but the difference did not achieve statistical significance (*p > . ). on other hand mean of sicam- , value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > . ). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < . ) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam- crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p- . . - protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr and increase in dcr expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type diabetes (t d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il- beta (il- b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il- b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit- mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il- b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at and h by mtt assay. trail ligand and receptor expression profiles on nit- cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit- beta cells at h, while increasing cell viability following il- /ifn/trail and il- /tnf/trail combined applications. substantial levels of death receptor (dr ) expression were detected on nit- cells before applications, yet it displayed decreased levels at h of trail treatment. lower levels of decoy receptor (dcr ) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il- b increased cell viability in nit- beta cell lines along with reduction in dr death receptor expression and an increase in the decoy receptor dcr expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u plus . microarrays, peripheral blood cell mrna profiles of bs patients (b) and matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of and for the chosen fold changes of . and . respectively (p ≤ . for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin- and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin- and homocysteine, we also investigated whether there is a correlation between these parameters. obese and healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin- and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c -esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c _ inh deficiency. materials and methods: people with c _ inh and people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss . was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = . ), looh (p = . ) and sh (p = . ). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: + . ) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, % fructose administered group, cape group, % fructose + cape administered group and ethanol group. after weeks, liver oxidant and antioxidant status, and blood tnf alpha, il- , and il- , tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il- , il- levels were found to be increased in fructose group compared with the control group (p < . ). in liver tissue of % fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul , n. uzun , e. c ß inar tanriverdi , h. € un agri ibrahim cecen university, agri, turkey, nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of pregnant women were included in this study. pregnant volunteers were divided into two groups ( healthy pregnant women and pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin- (il- ) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il- levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il- levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty -day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents hrs after lps injection. the rats were sacrificed at hrs after lps or normal saline injection (n = ). hepatic tissue tnf-a, il- , icam- and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il- , icam- and pdgf levels were statistically significantly higher than in controls (p < . ). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il- , and pdgf when compared to sepsis group (p < . ). there were no significant differences in the icam- levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il- , icam- and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of mg/kg during weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival ( %) compared to rats without treatment ( %). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel ( / nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red ( nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic doses of dtx, cis and deg. on the a and h nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v. . . ) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than . were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic values). these effects were remarkable in p -mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the % of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic doses of dtx, cddp and selenium on the a and h (p mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic concentrations at lower doses in both nsclc cells. however, cddprelated ic concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor (her ) is overexpressed in % of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her overexpression. in breast cancer cell line mcf , her overexpression (mcf-her ) results in mitochondrial dysfunction with low mitochondrial membrane potential (Δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf -her cells than vector control (mcf -vec). additionally, mcf -her cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf -her and decreases in knockdown of her in hcc cells that is her positive breast tumor cell line. based on these results, we conclude that there is a link between her overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed microrna genes deregulated by methylation and new potential target genes of mir- and mir- - p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with references (rnu , rnu ) and paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir- b- p, - - p, - - p, - a- p, - b- p, - - p, - - p, and - - p (p ≤ . , fisher's exact test) in bc. we observed mirna genes to be hypermethylated and mir- hypomethylated. hypermethylation for of these mirna genes was shown for the first time: mir- , - , and - ( - % of bc cases). a significant correlation between methylation and expression alterations was revealed for mirnas with downregulation: mir- b- p, - - p, - - p, - a- p, and - b- p (spearman's correlation coefficient (rs) was in the range À . to À . , p ≤ . ), and for mirnas with both scene (down-and upregulation) as well: mir- a- p, - a, and - (rs = À . to À . , p ≤ . ). comparative analysis of the data on expression alterations of mirna genes and protein-coding genes, which were predicted as targets by mirwalk . , revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir- /rhoa, mir- /rassf (a), mir- - p/dapk (rs = À to À . , p ≤ . ). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant - - from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: -year survival is about %. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of paired (tumor/ normal) ccrcc samples. we also used post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and paired ccrcc samples. we observed mirna genes (mir- a- /- /- , - - , - - , - b/c, - - , - a, - ) to be hypermethylated, (p ≤ . , fisher's exact test), mirna genes to be hypomethylated and mir- a with both scene (hyper-and hypomethylation was detected). methylation of mirna genes (mir- a- /- , - b/c, - - , - , - a, - a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir- - , mir- a, and mir- significantly correlated with metastasis presence (p < . , fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir- - and up-regulation of p protein-coding genes: rarb( ), rhoa, nkiras , and chl , which were predicted as targets by mirwalk . (spearman's correlation coefficients (r s ) was in the range . - . , p ≤ . ). in conclusion, novel supposed interactions of mir- - with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir- - , mir- a, and mir- could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm and eno genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset ( k illumina microarrays) we supposed that cpg methylation of pgm and eno promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm :~ bp and bp upstream tss; eno :~ bp downstream tss) that are frequently methylated in hc samples (up to %, / ) with down-regulated pgm and eno expression. thus, we demonstrated frequent and significant pgm and eno down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm and eno gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mК- . . from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of sprague dawley rats were randomly divided into six groups. rats were administered with mnng ( lg/ml) by oral gavage on days , and to initiate stomach cancer. during the experiment, rats died and those surviving were sacrificed in the rd, th, th, th and th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains rat which are given only food and water for months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows . , . , . , . and . for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < . ) and of groups iii, iv and v (p < . ) and also were lower than in the control group (p < . ). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: / ). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep- ), by considering its anticancer features. water extract of turkish propolis (wep, - mg/ml) and ethanolic extract of turkish propolis (eep, . - mg/ml) were prepared and incubated with hep- cell lines ( , , and h). mmp was investigated with a flourometric method by using dioc ( , -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep- cell series according to control ( concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf (nuclear factor ) family. there are few data suggesting that the nf factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf -b isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < . . these data suggest the involvement of the nf proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal ( ; ) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than % of chronic myeloid leukemia (cml) cases. epigallocathecin- -gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k- cells. the cytotoxic effect of egcg on k- cells was determined in time and dose dependent manner by wst- analysis. total rna was isolated from k- cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac , hdac , hdac gene that supports cell proliferation was down-regulated . , . , . folds in k- cells treated with ic dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. patients with cll and healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = . ) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = . ). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = . , p = . ). cut-off value of . absu for anti-ca i was associated with % sensitivity and % specificity and a cut-off value of . absu for anti-ca ii was associated with % sensitivity and % specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained noti-clones associated with chromosome genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, noti-sites with high ( - % of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh l , vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn , foxp , prickle , etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras , itga , lrrc b, and ctdspl genes. several genes with high hm/d frequency (aldh l , ephb , itga , and ropn ) were tested for expression alterations using qpcr. frequent ( - % of cases) and significant (> -fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh l geneon the average -fold mrna level decrease in % of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh l , ephb , itga , and ropn genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant - - from the russian science foundation. functional hypermethylation of a number of chromosome genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained noti-clones associated with chromosome genes, were used for comparative (tumor/normal) hybridization of dna from paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, noti-sites with % and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga ), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn , nbeal , and ube e ). the highest hm/d frequency was observed for ankrd , nkiras /rpl , itga , cmtm , and gor-asp /ttc a genes - - %. expression alterations were evaluated for genes with high hm/d frequency (plcl , prickle , and ppp r a) and significant mrna level decrease (> -fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl , prickle , and ppp r a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from % to % of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost %. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons - , which corresponds to a control group consisting of people living in the city of balkhash. on the whole, molecular genetic studies have shown that examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of individuals including patients and healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than . ). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than . ). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than . ). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately % of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy y cells was investigated by crystal violet staining. ec was found to be . %(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p mrna levels. on the other hand, bcl- , bcl l , erk- , jnk, caspase and mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p and pten remained unchanged. these results indicate that another effector caspase such as caspase or may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor (irf ) is upregulated in melanomas, and genome wide association studies linked variation at irf locus with skin cancers. irf was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf in various hematopoietic cancer types. the aim of this study is to investigate the role of irf in melanoma cell lines. lentiviral vectors were used to reduce irf levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf knockdown (gfp positive cells) over melanoma cells with normal irf levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf levels. cell cycle profiling showed increased g /g and decreased g /m levels in irf knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf levels. taken together, these results indicate that irf knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf . these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. , -dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, adult wistar male rats weighing - g were randomly selected and divided into eight groups. mg/kg body weight (b.wt.) morin and . mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with . mg/kg b.wt. dmba three times during the administration period ( days). cytochrome p a (cyp a) associated -ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were ae , ae , ae , ae , ae , ae , ae and ae pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were ae , ae , ae , ae , ae , ae , ae and ae nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad and smad by the type i receptor and formation of smad / / heteromeric complexes that enter the nucleus to regulate transcription. -deazaneplanocin a is an inhibitor of the histone methyltransferase ezh . we aimed to reveal the effect of -deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg cell line. hepg , a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with % fbs. the cells were seeded the day before -deazaneplanocin a administration and then the cells were treated with lm -deazaneplanocin a for days. expression levels of genes were analyzed by roche lightcyclerÒ . gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < . was accepted as statistically significant. -deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads - in hepg cells (p < . ). we also found that -deazaneplanocin induces apoptosis in treated cell line (p < . ). as a result, -deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf- compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > . /l. differential leukocyte counts were obtained by bc (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < . ). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = À . ; p = . ). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy y cells. ec was found to be . % (w/w) essential oil for sh-sy y cells. neuroblastoma cells were incubated at °c for h. after h, cells were harvested for further studies. bax and p mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl- , bcl l , casp- , casp- , erk- and jnk expression were significantly downregulated. unlike these proteins, p and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp- and casp- . additionally, erk and p mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy y cells. p- . . - the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment ( mg/kg b.w., i.p.), ng and ng-ox ( mg/kg b.w., i.p daily for days) alone treatment; cis + ng ( or mg/kg b.w., i.p daily for days) and cis+ng-ox ( or mg/kg b.w., i.p daily for days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over . million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at À . v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c. . . . ) with glutaraldehyde could be achieved over a wide range of relative mole ratios in mm phosphate buffer at ph . , glutaraldehyde concentration of % . . discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u cell line. changes of apoptosis and cell cycle in u cell line exposured to ic dose of astaxanthin ( . nm- lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u cell line to which astaxanthin was treated or not. ic dose of astaxanthin was calculated as . lm at h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of % in u cells treated by ic dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u cell line and astaxanthin caused an increase of g /m phase arrest ( . fold) and s phase arrest ( . fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g /m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor (irf ) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf expression levels are elevated in several lymphoid cancers, and irf is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf -linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf expression levels. however, there are no published studies about irf target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il- , tnf-a) in non-small cell lung cancer (nsclc). material and methods: patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi ; ifn-c, il- , tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c . post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = . , p = . , %ci; . - . ). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = . , %ci; . - . , p = . ). after crt, mean alt level was determined as . . there was survival difference in nsclc patients with high post-crt alt (hr = . , %ci; . - . , p = . ). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l ) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic concentrations for h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. -aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < . ) through both cells treated with c- and c- . only c- decreased methylation level on l but not on hela. consequently, c- and c- caused demethylation on hela cells similarly with -aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c- and c- might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il- b and il- , causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il- b signaling increases activity of nf-kb pathway. at the same time, il- , triggers jak-stat signaling pathway, which effector is stat . nf-kb and stat activity facilitates hyperexpression of mir-nas mir- , mir- and mir- as well as down-regulates expression of mirnas mir- / , mir- and let- . this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac / / / and sirt / genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir- , mir- or mir- . also, these mirnas can silence ezh , mll, mll , nsd , setd / / , smyd , suv h genes encoding histone methyltransferases. mirna mir- suppresses gene encoding de novo dna methyltransferase dnmt b. at the same time, down-regulation of mirna mir- / can allow hyperexpression of gene encoding acetyltransferase elp . these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd and bd ). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd -selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp , and ctdsp , plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp and ctdsp dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir- - - microrna cluster in co-regulation of ctdspl/ / genes in nsclc was predicted. the significant ( -fold on the average) and simultaneous decrease of mrna levels of ctdspl/ / genes was revealed in the majority of nsclc samples ( %, / ). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = . - . , p ≤ . ) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir- - - family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples ( %, / ) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir- - - micrornas in the regulation of rb protein activity via inactivation of ctdspl/ / in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for were reviewed. for , a total of patients and tumor marker requests were included. carbohydrate antigen - , cancer antigen , cancer antigen - , prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of , both positive tumor markertest resultsratio and the positive patient ratio were %. in the patients group with increased marker levels, % of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, % patients with diagnosed cancer history in remission. the ratios of positive tumor markers were % forca - , % for ca , . % for psa, %for ca - , . %for afp, and . % for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct colon carcinoma cells. ic doses of resveratrol were determined by wst- assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd , fra , ppard, egfr, birc , pcna, mcl , stat , fos, jun, p , atf , trail, puma, gadd a, rb , faslg, tnf, socs , stat gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol ( lm) causes decrease in cell viability and increase in apoptotic cell death in hct p (+/+) and hct p (À/À) cells significantly (p < . ). the fold changes of the gene expressions have shown that resveratrol has significant (p < . ) and different effects on the expressions of the genes related with the existence of p in hct cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group and group , respectively. hypermethylation was detected in atm, cdkn a, and gata genes. mortality rate was ( . %) in group and group (p > . ). mean -years survival rate in group was ae months and mean -years survival in group was ae months (p > . ). positive median lymph node count was ae for group and ae for group and the difference was statistically significant (p < . ). frequencies of perineural invasion and lymphovascular invasion rate in two groups were % (p > . ). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for % of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p mutation, which is mutated % of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p (+/+) and p (À/À) hct colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate ( mm) and disodium pentaborate decahydrate ( mm) significantly causes nearly % reduction of cell viability at h (p < . ). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi k/akt, map kinases and many epigenetic regulators. satb is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb expression in nsclc cells. we used a and h cells as a model to evaluate the effects of the crosstalk between p and akt on emt of nsclc cells. therefore, cell culture, inhibition of p activation via sb , transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb , wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb , p-p , p , p-akt and akt was examined in a and h cells by western blot analysis. we observed that e-cadherin and satb are downregulated in a cells (highly active p , lowly active akt) compared to h cells (lowly active p , highly active akt), suggesting that e-cadherin and satb are associated with the crosstalk between p and akt pathways. our results demonstrated that p inhibition in a cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p inhibition upregulated satb expression, and reversed emt in a cells. furthermore, alone satb knockdown is sufficient to induce emt, and prevented the effects of p inhibition on emt. all these results strongly indicate that the crosstalk between p and akt pathways might determine satb expression and epithelial characters of nsclc cells, and satb is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p and akt signalling pathways could regulate satb expression. this work was supported by tubitak ( s , z ). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron vntr and exon -g t (glu asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. ) and bioenergetics (pallotta ) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d -pyrroline- -carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put p and put p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta ) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put p/put p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group ), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dΨ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. (pallotta et al. , (pallotta et al. , pallotta ; di martino pallotta ) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : patients who were diagnosed with colorectal cancer between january and january ; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was . . cancer-specific survival was not significantly different between the high-and low-lmr groups (p = . ). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: . ; % confidence interval: . - . ; p = . ). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment and treatment . . ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment groups, and the treatment group were given mg/kg and mg/kg dose of luteolin, intraperitoneally, for a days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < . ) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < . ). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around - % of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu- , snu- and snu- than the well-differentiated hcc cell lines such as huh- , hepg and plc/prf/ . besides, expression of txnip was examined in non-hcc and hcc tissue samples by immunohistochemical staining. txnip positivity was observed in % of well and % of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk- b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak kinase (ag- ) on myocardial infarct size and the jak /stat signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia ( % o , weeks, h/day) and part of them recieved ag- ( mg/kg) min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak /stat signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak inhibitor ag- caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak , stat and some other proteins involved in cardioprotection (akt, erk / , gsk b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p- . . - detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection ( mg/kg, twice a week) following the unilateral nephrectomy in male -week-old wistar albino rats for weeks. blood pressure was measured by using tail-cuff method. gw ( mg/kg/day, i.p.) was administered last week. expression of various markers (grp , perk, p-perk, ikb-a, nf-kb p , tnf-a, bax, bcl- , mmp- ) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp and p-perk were increased by hypertension and gw treatment reversed them. hypertension-induced increase in nuclear nf-kb p expression and decrease in ikb-a expression were reversed by gw treatment. while bcl- expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp- expression was augmented and gw treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw treatment. these results suggest that lxr activation by gw restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph ( . mg/kg/day or mg/ kg/day, per os) from p -p . chronic mph treatment ( mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin- and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin- and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin- , with no alterations in claudin- . overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs directly interacts with and interferes in opioid receptor (or) signaling. rgs is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs can regulate transcriptional responses mediated by the stat b transcription factor. isolated neural stem cells from rgs À/À mice were immunostained for the mitosis marker ph and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro a cells stably expressing rgs . the functional assays of stat b transcriptional activation were performed in hek expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs blocks stat b phosphorylation and transcriptional activation by interfering in stat b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs results in increased mrna levels of stat b target genes such as the members of the bcl anti-apoptotic family bcl- , bcl- and bcl-xl. this upregulation of stat b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs in stat b mediated transcriptional responses and a novel selective role of rgs in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph - molten globule structures was formed while the pre-molten form took place under ph . in our tests at ph . when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph . in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [ , ] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel- (vdac- ), mitochondrial-plasma membrane cofilin and caspase- activity was observed in the hippocampus of ka treated rats. administration of mi h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca and ca subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references . r. . neuroscience letters, vol. , no. , pp. - . . r. solomonia, et al; . cell. mol. neurobiol. vol. , no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic values of . lm and . lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab / . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin- (ps ) with - mm azure b or vehicle for h. to determine the effect of azure b treatment on ab / levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab and ab levels with a dose range treatment of azure b in ps cells. ab levels were reduced by . % in lm and . % in lm azure b-treated cells when compared to control. additionally, ab levels were decreased by % in lm and . % in lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy y) used as a tumoral model or differentiated to a neuronal phenotype. pink role in this process was analyzed using genetically engineered pink deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p- . . - intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment ( - ) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage ( - ) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal - dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included female day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during weeks. the rats in the exercise group swam for weeks. after weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function ( ) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain ( ) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad ( ). we initiated a study to screen their acetylcholinesterase (ache, ec . . . ) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic values in the range of . - . nm for ache. this study was financed by turkish research council-tubi-tak (kbag z ). p- . . - f -a h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut , a. sen , d. doyduk , y. yildirir department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, gazi university, ankara, turkey fty , a sphingosine -phosphate (s p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty , namely f -a h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f -a h along with fty using pgl . luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty treatment at non-toxic doses. it was found that f -a h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty does. furthermore, f -a h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f -a h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab - ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a nicotinic acetylcholine receptor (a - nachr) domain resulted in astrogliosis, decrease of a nachr density, accumulation of ab - in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c bl/ mice were injected with lps, immunized with recombinant a - or a - endoglycosidase treated to remove carbohydrates. two immunizations with week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a - -specific antibodies and brain preparations were studied for a nachr, ab - and il- levels. the original a - ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a - immunization with 'deglyc' a - did not stimulate il- elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a resulted in similar a nachrs decrease and ab - accumulation in brain and significant episodic memory decline comparable to those after lps injections. a nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a nachr density caused by a - -specific antibody is critical for ab - accumulation and episodic memory impairment while pro-inflammatory capacity of a - -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using , -diphenyl- -picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl , the cxcr ligand. we aimed to determine whether snp rs located in the promoter of cxcr gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr promoter activity. mef c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr promoters containing different rs alleles were estimated using luciferase reporter assay. we determined that minor rs allele creates functional mef c-binding site within one of the regions required for the basal activity of the cxcr promoter. cxcr promoter containing minor rs variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef c as the main regulator of rs -dependent modulation of cxcr promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d receptors (d r) and histamine h receptors (h r) form heteromers through which h r ligands can inhibit d r function. d rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d r and h r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d r and h r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk / phosphorylation and cell death in cortical slices. endogenous d r-h r heteromers were detected in rat and mouse cortical slices, where h r ligands decreased d r signaling (erk / pathway) and were also able to block the cell death induced by overstimulation of either d r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d r and h r form heteromers with nmdar subunit a in the presence of subunit b. d r-h r-nmdar heteromers were detected by bret with bifc. endogenous d r-h r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d r, h r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d r-h r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h r ligands act as a 'molecular brake' for d r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with weeks old total male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for s to co and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at À °c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi- and cd . changes in tight junction (tj) proteins (occluding and zone occludin- ) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi- staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec . . . ) is a zinc metalloenzyme that catalyzes the reversible reactions of co and water. carbonic anhydrases (cas, ec . . . ) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose- b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained . m tris-so ph . , r-nitro phenol in ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: rats were used. for sit, animals were kept individually for month and for pst, rats were exposed to dirty cat litter for min at the first day of month stress. flu was given ( mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < . were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < . ). flu restored anxiety scores in both sexes (p < . ) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < . ). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < . ) and pst (p < . ) in both sexes. female rats had % lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < . ). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein (hapln ) is one of the major elements of pnns. hapln interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln were observed. however, the role of hapln in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln during differentiation using pc cell line as a neural differentation model, derived from rat pheochromocytoma. after pc cells were stimulated to differentiate into neurons by nerve growth factor on days , and ; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln and survival motor protein (smn ) network analysis were performed in-silico. as a result, we analyzed fold increase in hapln mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln level suggests that, hapln is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln and smn through fibulin (fbln ) was detected. fbln was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln and fbln in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf . the risk of malignancy is increased in nf , wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf , we considered the mesenchymal stem/stromal cells (msc) carrying the nf mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf -mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen . mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of g) chronically intoxicated with hepatotrophic toxine of taa. every weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by % in the experimental group. in the experimental hepatic x-amidase activity decreased by % compared to those in the control. the average x-amidase activity in the blood serum ( . nmols/ mg/min) and in the brain ( . nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about % higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached %. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi x ). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp b gene. atp b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t i, p.v i and p.r g-fsx in yeast lacking the ccc gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t i mutation mainly affects the liver and the nervous system, p.v i mutation affects the nervous system, and p.r g-fsx mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp b, we cloned human atp b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp b genes were transformed into yeast cells lacking the homologous ccc gene for functional comparison. first, we analyzed the expression of atp b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp b gene in ccc d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t i, p.v i and p.r g-fsx mutations cause functional deficiency in atp b functions and suggest that these residues are important for normal atp b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: z ). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen , , e. erem faculty of medicine, ege university, izmir, turkey, center for continuing education, ege university, izmir, turkey, recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. students (in rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was . / , that increased to . / in the posttest evaluation. % of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was % in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates ( , , lg/well) and lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by % sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < . was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv . voltage-gated potassium channel kv . belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv - subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s -s linker at the inner mouth of the pore. here we present an improved d structure of the truncated human kv . channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv . channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag channel, full-length shaker potassium channel kv . , c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a -year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of graduation. currently, there are students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube : z (becton dickinson and company, franklin lakes, nj, usa); tube : ayset (lot , turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au ) and randomised samples stored at - °c for and h. th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at , and h were statistically no significant (p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' ( / / - / / ) (search # ). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search # ) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search # revealed articles; of these were clinical trials, were observational studies. human studies were while animal studies were . overall, there were review articles and meta-analysis in the field. search # revealed articles of which were clinical trials, were review articles, were meta-analysis. human studies were and other animal studies were . the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi kinase, pi-dependent kinase and akt. then, we found the d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopÒ software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of ptb ( s, msc, phd theses) . % was in natural sciences while . % was in health sciences. the theses output-growth measured by the compund annual growth rate was % over the -years. the top clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants ( ), endocrine-metabolism ( ) and enzymology ( ) were the top research areas in ptb, followed by genetics ( ) and cancer ( ). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february , organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for days was realized with participants in total, of which from universities; representatives of their companies; from chamber-institute-public establishments and of which from public hospitals. of the participants were from izmir, of them were coming from out of izmir. conclusion: at the end of the symposium, % of the participants gave feedback. among the feedback selected; . % of the participants evaluated the symposium overall, as successful. . % of them found the symposium successful with regard to its scientific content. their feedback were . % positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and . % of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a item online survey with sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the -day period, ms students (in the thesis phase) answered the questionnaire (female: . %, male: . %). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work ( %), visits with their mentors ( %) and theoretical lessons ( %). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only % and % sufficiency, respectively). communication skills ( %) and team work ( %) were rated as the highest professional competencies followed by literature search and research planning (both %). it was striking that ms perceived themselves as quite insufficient in scientific writing ( %), data analysis ( %) and project writing ( %) while proficiency in english ( %) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority ( %) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu- phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached phd graduates who had graduated from deu graduate school of health sciences between and from different departments via e-mail. the survey was included questions, which were prepared in the light of the existing literature. among the phd graduates, ( %) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (% . ) are well satisficed from the education they had taken. beside this, interestingly we found that % . of graduates prefers staying in the academic positions and % . of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn and itsn are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using web servers we analyzed utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir- , mir- , mir- , mir- / , mir- , mir- and mir- in utr of itsn -s, predicted by servers, mir- predicted by servers for itsn -l, and mir- , mir- / , mir- , mir- and mir- predicted by - servers for itsn -l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir- , mir- , mir- / , mir- , mir- and mir- were highly enriched for all analyzed pathways. using regrna . and scan for motifs services we predicted types of different regulatory elements in utr of itsn and itsn : k-box and brd-box, musashi binding element for rbps musashi and musashi , gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn -s regulation by micrornas we cloned utr of itsn -s into luciferase reporter vector and transfect hek cells by this construction and mir- a, mir- a and mir- a. for mir- transfected cells, we found - % decrease of expression of itsn -s utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir- target site in utr of itsn -s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. % of stroked patients lose their life in year, and remaining / of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir- a, - , - , b, - , , - a and let- f of ischemic stroke patients and to investigate role in predicting prognosis methods: patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and healthy individual were included in the study. after stroke patients' blood samples were taken periodically in st day, st week, and rd month, and at the same time nihss and mrs scores were determined. set mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke st day peripheric blood levels of mir- a,- ,- a and let- f were significantly low; when st week and st day serum records were compared there was a significant increase in mir- level; and when st week and rd month records were compared we noted that there was a significant increase in mir- a,- and let- f levels. from prognosis point of view; after ischemic stroke measurements showed that mir- b in the st day, mir- a and mir- in the st week showed positive significant correlation with rd month mrs scores (p = . , p = . , p = . , respectively) conclusion: according to the outcomes of this study, after stroke st day mir- b, st week mir- a and st week mir- levels can be stipulated to use in predicting patients' rd month prognosis p- . . - inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [ ] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing -nucleotides of a random region. after rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that -fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [ ] hunsicker, et al. ( ) an rna aptamer that induces transcription. chem. biol., : - . p- . . - microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyÒ mini-kit. the expression profiles of mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir- - p was expressed only in aorta. mir- - p and were expressed both aorta and whole blood. mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > ). mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir- - p, mir- - p and might be a potential for cardiovascular disease development. also mir- - p and might serve as novel non-invasive biomarkers for cad p- . . - mir b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc- and miapaca- cells transfected with mir b mimic. our data revealed that mir b led to decrease the cell viability depending on enhanced mir b doses, which are , , and nm, as the ratio of % , , and , respectively, in miapaca- cells and as the ratio of % , , and , respectively, in panc- cells compared with control condition of each cell. this inhibition mediated by mir b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir b was more effective than gemcitabine. based on our findings, it is clearly shown that mir b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant s . breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her + breast cancers, which has role in approximately % of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her + breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt and skbr cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that out of the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t( ; ) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k cells were treated with lm (ic dose) res during h and cytotoxicity was evaluated by using wst- assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir- - p level . fold and significantly downregulated hdac gene expression (p = . ) and upregulated p / sqsmt gene expression (p = . ), according to the control cells.p /sqstm interacts with lc and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac and p /sqsmt gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac dependent induction of gene associated with autophagy like p /sqsmt and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir- , hsa-let- a, hsa-mir- b and hsa-mir- . thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let + , let + b, let + , + b, + , + let + b, + let + b. the cell viability was detected by wst- kit in a well plate elisa reader. cells were seeded as per well, mirnas incubated with cells for h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however + b and + combination increased the cell viability dramatically. on the other hand let + b and only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein (sip ), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip cernas listed at competive endogenous mrna database (cerdb), transcripts (pten, zeb , ptch , creb , acvr b, enah, robo , erbb , e f , foxo , rictor, klf , ets , cdk ) sharing at least common mre sites with sip were selected and their expression in hcc cell lines were determined by qrt-pcr. ets was found to be highly correlated with sip in hcc. furthermore, repressing sip expression by shrna in hcc cells resulted in decreased expression of ets , pten and zeb . our results suggest a possible bidirectional and post-transcriptional regulation of sip and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar , , n. belder, h. ozdag biotechnology institute, ankara university, ankara, turkey, comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of crc tumors against normal colorectal mucosa samples, using raw data from e-mtab- and e-geod- (affymetrix microarrays), and gse and e-mtab- (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ . , fold change ≥ . and stringent false discovery rates. mirtarbase and mirwalk . databases were explored to identify validated targets. we found thirty (including mir- and mir- ) and thirteen (mir- , mir- , mir- , etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol- kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan , h. g€ urdal , b. g€ ur dedeoglu biotechnology institute, ankara university, ankara, turkey, department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip ( , , ) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip is generated by hydrolysis of pip ( , ) bisphosphate, which is synthesized by pip k . any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt- was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt- cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip k c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk- -beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl- , crl- , crl- , crl- and htb- ) were evaluated with non-malignant (normal) brain cell line (hcn- ) . determinations of expression level of five mirnas (mir- , mir- , mir- , mir- , and mir- ) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir- , mir- , mir- , and mir- ) were significantly decreased while the expression level of mir- was increased in gbm cell lines according to hcn- cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v . , mir disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn b-as, locus cdkn a/b (ink /arf), chromosome p ) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan . , we predicted target sites for hsa-mir- -p/ -p/ -p/ -p/ -p/ -p and hsa-mir- - p/ in anril utr. then, we used hmdd v . and mir disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir- is implicated to alzheimer disease and mir- to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir- and mir- targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril utr. conclusions: we predicted sites for mir- and mir- in utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different ( . - mg/ml) concentrations for h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir- b- p (- , fold, p = . ), hsamir- - p (- , fold, p = . ), hsa-mir- a- p (- , fold p = . ), hsa-mir- a- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- b- p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- a- p (- , fold, p = . ) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir- b- p plays a critical role in the matrine response. ericd (e f -regulated inhibitor of cell death) is a newly found lncrna. it is located at q . . it has two exons and its transcript size is bp. ericd is regulated by e f (transcription factor ) and modulates the cellular response to dna damage. arid a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, different cancer cell lines, osteoblast cell line and different types of normal human tissues rnas were selected for expression analysis of ericd and arid a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid a had lower expression in u- os (osteosarcoma), a- (glioblastoma) and a (lung cancer). at the same time, ericd expression was lower and arid a had high expression in hfob . (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid a are cell cycle regulated and are commonly regulated by e f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif a and anti-eif c. mir- - p, mir- a, mir- b, mir- - p, mir- , mir- a*, mir- , mir- a, mir- a, mir- b, mir- a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif c immunoreactivitiy was negative in all groups. mir- , mir- a* and mir- a were highly expressed in proliferation phase of fertile group, mir- a and mir- b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over . million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately nucleotide length which regulates the expression of genes by binding to the -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed unique small rna sequences obtained from raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified putative mature microrna sequences belonging to distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. of precursor sequences showing % homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as , a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as . materials and method: for the tissue expressions analysis, human total rna master panel ii (containing different human tissue samples) was used. total number cancer cell lines and normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as were found to be tissue specific. particularly, while vim-as was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as was also found to be differentially expressed in cancer cell lines. vim-as expression was found to be lost in cal , pc , and hct and highly diminished a cancer cells. also, it is found to be highly expressed in bcpap, panc and beas b cells. discussion: results of the current study suggest that vim-as may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as and vim axis can be involved in the formation of lung tumors because vim-as was highly expressed in normal lung tissues and beas b cells and expressed very low levels in a lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately % patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h and pc cells treated by tnf-a ( ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir- a- p, mir- as- p, mir- , mir- , mir- - p, mir- q mimics can induce cellular invasion on h , mir- v, mir- h- p, mir- - p, mir- a- p, mir- as- p, mir- mimics can induce cellular invasion on pc . we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir- q, mir- a- p, mir- as- p, mir- , mir- in h , mir- - p, mir- a- p, mir- as- p, mir- in pc can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir- a- p, mir- as- p and mir- . (this study is supported by tub _ itak grand number s ). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a cells. when we inhibit nf-kb activity by p targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir- as and pten untranslated region has recognition site for mir- as. therefore; we transfected a cells by mir- as. mir- as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir- as. (this study is supported by tubitak grand nummber s ). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a (autophagy and v-atpase inhibitor, . pg/ . ml/ovary) on day of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc -i was not significantly altered throughout pregnancy, that of autophagosome-associated lc -ii increased significantly from day , showed a peak on day , and decreased on day (day of normal parturition). lc -ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day . in vivo treatment with bafilomycin a resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc -ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in . the value of low dose bpa was received as < lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i- therapy, have been examined in patients ( m/ f) with ptc and ( m/ f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at days after i- administration ( . gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp- , mmp- , timp- and timp- were measured by elisa. there were no significant changes in serum concentrations of mmp- , timp- and mmp- /timp- ratio after i- in the two groups. in ptc patients, i- administration resulted in an increase with % in timp- level (p = . ) and a reduction with % in mmp- /timp- ratio (p = . ). in ptc+ht patients it has been observed an increase with % in tgab level (p = . ), % in mmp- /timp- ratio (p = . ) and unchanged timp- serum concentration. tgab titers were positively correlated with mmp- /timp- ratio (r = . , p < . ). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp- /timp- for anti-invasion and anti-migration by augmenting timp- . in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts , and and mmp , and to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, cases have been included in this study, divided into groups according to their pmis: ' - h', ' - h' and ' - h'. from these tissues, western blotting technique is used to analyse the expression of adamts , and and mmp , and . it is determined that when pmi increases; adamts- and amounts decrease. on the other hand adamts- amounts are examined to increased related to the interval., especially on the ' - h' dataset. additionally, considering metalloprotease levels, mmp- and amounts decrease as pmi increases. unlike mmp- and ; mmp- levels increase proportional to the interval, especially on the ' - h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts- and mmp- , proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases and in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp and mmp (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp - c>t and - c>t polymorphisms cause loss of gene function, and mmp - c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp - c>t and - c>t polymorphisms, and mmp - c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from healthy subjects. the risk of abortion was . -fold higher in patients with heterozygous - c>t polymorphism (p = . ). combined genotype analysis showed that the risk of abortion was . -fold higher for patients with normal - c>t polymorphism, and heterozygous - c>t polymorphism (p = . ). functional polymorphisms of mmp and mmp may have a role in etiology of abortion. p- . . - changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses ( . and mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, min and min ischemia-reperfusion. after months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts- and - genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam , - , and - . in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = ), oligozoospermia (os; n = ), azoospermia (as; n = ) groups. adam , - and - protein levels in infertility indviduals were analysed by western blot. adam protein level was . fold lower in the os and as groups than in the ns group. adam protein level was . fold higher in the as group than in the ns group. adam protein level was fold lower in the as group accourding to ns group. we observed no change between protein level of adam and adam of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin- on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck- , timp- and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created patients with diagnosis of cchf. healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc- and timp- levels of patients and a control group were analyzed using the human elisa kits. serum timp- tck- and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp- , tck- and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p- . . - activation of calpain and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase (mmp ) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca + homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine in gene of cystic fibrosis transmembrane conductance regulator (f del-cftr), could be involved in the abnormal presence of mmp in the extracellular fluids of cf patients. pbmcs were isolated from healthy donors and cf patients homozygous for f del-cftr. mmp levels were evaluated following h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp ; conversely, in pbmcs from healthy donors, expression and secretion of mmp are undetectable but both events can be evoked, after h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and h-cultured control pbmcs mmp secretion is prevented by chelation of intracellular ca + and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases and (erk / ). moreover, the rescue of active f del-cftr reduces the extent of mmp secretion, correlating the channel defect to the [ca + ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca + concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp . these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp in fluids of cf patients. p- . . - aebp /aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir , , u. akpulat , i. onbasilar , c ß . kocaefe department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, department of stem cell, institute of health, hacettepe university, ankara, turkey, laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp /aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp is the short isoform that acts as a transcriptional repressor by targeting the ap promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp /aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp /aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp /aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated folds upon the induction of differentiation in c c cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp /aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp /aclp expression peaks up to folds in the th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp /aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp /aclp expression is upregulated up to folds in early time-point samples. these results depict a novel role for aebp /aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus - encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus - extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average % compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a -fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo a remained at the level with expression in the strain with the complete spo a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo b, spo f, spo k, spo j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p- . . - paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for min followed by h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp- and - were performed after h of iri. the effects of paricalcitol on activities and expressions of mmp- and mmp- levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp- and mmp- during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp- and - . p- . . - the changes of matrix metalloproteinase , activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko , o. shaulska , y. kot , g. ushakova , a. shevtsova dnipropetrovsk medical academy, dnipropetrovsk, ukraine, kharkiv national university, kharkiv, ukraine, dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp and mmp activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the wister rats divided to groups were used for the experiment. cdcl x . h o in doses . lg/kg and lg/kg was given to rats intragastrically in drinking water during days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp and mmp , total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium ( . lg/ kg) lead to reduced activity of pro-and active forms of mmp in myocardium ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly) and in serum ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly), but pro-mmp activity in heart was increased ( . ae . au/mgtp compare to the . ae . au/mgtp in the control rats); level of ha was decreased in both tissues ( . ae . lg/ml and . ae . lg/ml compare to the . ae . lg/ml and . ae . lg/ml in the control rats accordingly). high doses of cadmium ( lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp increased from . ae . au/mgtp to . ae . au/mgtp, prommp to . ae . au/mgtp, mmp to . ae . au/mgtp. ha level was increased in serum ( . ae . lg/ml) and decreased in heart ( . ae . lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin- repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin- (il- ) is an il- cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il- implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts in u proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of lg/ml while trypsin and chimotrypsin are used as - mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no - - ). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from to lmol resv and opposite effect from to lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn- . since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in . m kcl (ph . ) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments - of amyloid peptide, fragment - of [gly ]-amyloid peptide and - of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs ( - - ) and sp- . . . ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti , c. bernacchioni , f. scaletti , p. turano cerm (magnetic resonance centre), florence, italy, department of biochemical sciences, university of florence, florence, italy, chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort , t. ozben , a. sansone , s. barata-vallejo , c. chatgilialoglu , c. ferreri sanko university, gaziantep, turkey, akdeniz university, antalya, turkey, cnr, bologna, italy, universidad de buenos aires, buenos aires, argentina, national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at °c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr in glucose homeostasis. drugs targeting gpr may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr inhibition. in addition, gpr takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon and an additional trans double bond between carbons and . dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical -carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from to mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p- . . - trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b -arrestin or gb subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with À m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p- . . - l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev , , v. polovinkin , , , v. shevchenko , , v. gordeliy , , moscow institute of physics and technology, dolgoprudniy, russia, research centre j€ ulich, j€ ulich, germany, institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr has an absorption maximum at nm at ph . and to create more redshifted variants we analyzed available structures of the kr (pdb codes: xtl, xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m , g and s ). the mutants of kr under investigation were: m a, g v, m a/g v, s a, s f, s g, s l, s m, s n, s t, s v, s y. the protein mutants were expressed in escherichia coli c strain, expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside. the cells were then washed trice with unbuffered mm nacl or kcl solution. finally, the ph changes in cell suspensions (od = . ) were monitored. ph changes upon the addition of lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s f, s l, s m, s n, s v. the remaining mutants have shown sodium pumping activity and s a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph . ( mm na/na-pi, mm nacl). m a mutant absorption maximum is blue-shifted to nm. g v and m a/g vblue-shift to nm. s a, a potassium pumping variant,red-shift to nm. s g, s yred-shift to nm. s tno change in absorption maximum position. based on structural analysis of kr we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to nm and red-shift up to nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than % drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins - , which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. - - ). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv- ). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in biopsies ( %). c iv deficiency was most common ( %), followed by c i ( %) and c ii-iii ( %). multiple complex deficiency was present in % and isolated deficiency was present in % of the biopsies ( % c i, % c ii-iii, % c iv). cs activity was elevated in % of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in %, % and %, respectively. comparing with histological data, biochemical analysis revealed additional findings in % of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) , resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and groups were included in the study. rats were fed with chows that were given either normal diet for control group ( % carbohydrate, % protein and % fat), high fructose ( % carbohydrate ( % fructose and % starch), % protein and % fat), or high sucrose ( % carbohydrate ( % sucrose and % starch), % protein and % fat). rats were fed with chows for weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba c, glucose, resistin and adiponectin were studied. glut and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < . ). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < . ). there was no significant differance level of hba c, glucose, resistin and glut in rats fed sucrose or hfcs (p > . ). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p- . . - nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy , n. eruygur , e. s€ uleymanoglu biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg +) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg +-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group ). secondly mice were randomly divided in four groups as: group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days, group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days. ozone/oxygen mixture and oxygen were given at a dose of mg/ kg groups ( ) ( ) ( ) ( ) . after treatment animals were sacrificed, and muscle samples were taken and stored in À °c for until the analyses. muscle tissues were homogenized in . m tris-hci and . m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). sdh activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). there was no statistically significant difference among all groups in terms of cs (p > . ) and sdh activities (p > . ). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than . %, so they were scored as . there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of -monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of -monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, -( -{-[ -(octyloxy) phenyl] diazenyl} phenoxy) propane- , -diol ( % mol), was incorporated into the -monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn m phase with lattice parameter . angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at nm we observed trans-cis-isomerization. in the same time the cubic-pn m phase was not changed but the lattice parameter reduced to . angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin , t. cora cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt that is a hnscc tumor cell line and hek- that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf- r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of women ( %), and men ( %). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant . times increase of gpx activity levels; and . times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by . times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than . times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity ( mm) and potential protective effect of different doses of boric acid (ba) ( , and mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+ mm ba, ethanol+ mm ba, ethanol+ mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < . ). however the mda level in the ethanol+ boric acid ( mm) group was shown to be significantly decreased compared with that in the ethanol group (p < . ). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba ( mm, mm) groups were close compared with control groups (p < . ). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid ( mm) groups were increased (p < . ). these results demonstrate that ethanol ( mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co /bicarbonate between metabolizing tissues and lungs, ph and co homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = . ), (p = . ), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = . , p = . ). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = . , p = . ), (r = . , p = . ), respectively. at an anti-ca i cut-off point of . absu, sensitivity was % and specificity %. at an anti-ca ii cut-off point of . absu, sensitivity was % and specificity %. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from healthy control and patients with behc ßet's disease. the mean age for controls and patients were ae . and ae , respectively (p = . ). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v . results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ). lymphocyte counts were lower in patients with behc ßet's disease compared to control group ( . ae . vs. . ae . ) (p = . ). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of neuroblastoma patients ( male, female, median age: months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of healthy children. patients had stage , , or s disease, patients had stage disease. different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir ds (p = . ), and higher expression of inhibitory kir gene dl (p = . ). additionally kir ds genes were more common in patients with early stages (stage , , or s) (p = . ) and kir dl genes were more common in patients with stage (p = . ). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir dl gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir ds can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p- . . - neuroprotective and immunomodulatory effects of urtica urens s. arslan , g. terzioglu , b. kabalay , a. r. tufekci , a. sen , i. demirtas department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco- cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine (cxcl ), and (cxcl ) were determined. whole genome transcriptome analysis was performed by using human ht- v beadchip. extract treatment caused % and % increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly ( % and %, respectively). similarly, cxcl and cxcl mrna levels decreased % and %. transcriptome analysis showed that probes were significantly changed (p < . ). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak t . p- . . - linear low molecular weight a- , -glucan from bifidobacterium bifidum bim b- d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, , ( ) : - ) that bifidobacterium bifidum bim b- d contains low molecular mass ( - kda) a- , glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b- d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, h and c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as - kda linear a- , -glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b- d cells contain linear low molecular mass a- , -glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a- , -glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba c, uric acid, creatinine, ggt, -hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > . ) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < . ). on the other hand, rdw are determined to be significantly lower (p < . ) in the non-diabetic obese group. no difference was detected in -hydroxyvitamin d levels between the control and the study groups (p > . ). non-diabetic obese patients had significantly (p < . ) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < . ). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco , which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco is also known to be present on autophagosomes, and overexpression of fyco can induce the formation of enlarged lc -containing autophagocytic structures. here we demonstrate that fyco knock-down leads to defects in autophagic mb degradation, and that fyco functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p- . . - proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d and b ) and cell cycle controller genes (p and p ). sf cells were treated with different doses ( nm- lm) of hypericine for h and h. a significant cell proliferation was observed in moderate concentrations ( . - lm; % -% ), but at high concentrations ( - lm) cytotoxic effects emerged in sf cells (ic = . m, r = . ). qrt-pcr results revealed that the most proliferative dose of hypericine ( lm) stimulates cyclin d . the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b mrna, whereas it induced expression of p and p genes, and thus apoptosis was observed by dna laddering at the same dose ( lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately months, increasing to months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant, the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f -targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p- . . - leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t( ; )(q ;q ) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k subclones (k -ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k -ir cells are not only resistant to imatinib but also to nd, rd generation tyrosine kinase inhibitors. we demonstrated that k -ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k -ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k -ir cells forms intact tumor spheroids in d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k -ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd + cscs isolated from human osteosarcoma cell line saos- using macs magnetic separation technique were characterized, and their stemness properties under hypoxic ( % o ) and normoxic ( % o ) conditions were compared in two and three dimensional culture conditions. two different d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct / , nanog and sox compared to normoxic conditions in d cultures. although similar effects of hypoxia were observed in d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on d compared to d culture systems. this study is seen as an introduction to develop a more relevant d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd + cscs were isolated from human pca pc cells using a magnetic-activated cell sorting system. pc and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for h. apigenin dose dependently inhibited cscs and pc cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p and p (kip ). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases- , - and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf- in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase- while caspase- , tnf-a and bcl- levels remained unchanged in pc cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp- and - exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi k/akt and nf-kb p / p were significantly decreased after h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. s ). (pi k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi k activity contribute to phenotypic variability. our aim is to characterise pik ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi k activation on downstream output specificity in the cell. we are currently employing crispr/cas mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p a, allowing precise spatiotemporal control over pi k signaling to unravel the extent to which pi kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi k signalling and potentially aid the development of targeted therapies for human diseases of pi k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf as transcription factors, egfr and cdk as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥ ), prostate (≥ ), and colorectal (≥ ) cancer based on assessment of . ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. . ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck , ck ) and the leukocyte-specific marker as anti-cd . all samples were analyzed by bd facs aria iii flow cytometry. in total, patients and healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but - ctcs were detected with ck , , , -based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at °c with % co and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf- and mda-mb- cells. this effect was observed more prominent on mcf- . considering prfe therapy this effect is much more pronounced especially for mda-mb- comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut , glut , glut proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut , glut and glut proteins was detected in hamscs by immunofluorescence. glut , glut , glut protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd , cd , cd , cd (mesenchymal stem cell markers) and hematopoietic markers cd , cd b, cd , cd and hla-dr were negative. the presence of glut , glut , glut proteins and genes were identified in hamscs. in this study, for the first time in literature, glut , glut and glut gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) cl ] on mammospheres obtained from mcf- human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase / activity, annexin-v-fitc and bcl- activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc- staining. mammospheres exhibited increased oct- and sox (stemness markers) expressions compared to parental mcf- cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion ( . - lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase / activity, annexin-v-fitc positivity and bcl- inactivation. apoptosis was also confirmed with increased tnfrsf a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [ ] . in the development of primary cancers, the head and neck cancer is accounting for approximately . new cases annually around the world [ ] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [ ] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than % of deaths in cancer patients are attributed to metastasis [ ] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [ ] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [ ] . in our study, . ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck , ck for positive selection and cd for negative selection [ ] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level . one of the general methods in ctc detection is flow cytometry . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy , . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which . ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck , ck for positive selection and cd for negative selection, were analyzed by bd facs aria iii flow cytometry . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p- . . - determination of effect cytotoxic, apoptotic, caspace- activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations ( . , . , . , . , , , lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase- activity in breast cancer cells (mda-mb- , mcf , bt- , sk-br ) and normal cell (mcf a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp- , bcl- , bax, birc- , gapdh, caspase- , caspase- , caspase- , caspase- ) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < . ) effect cytotoxic, apoptotic, caspase- activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl- ) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd + gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the gbm cell lines (u , u , u and t ). cd + and cd À cells were seperated by macs method via anti-cd (ac ) antibody from cultured cells and cell lines. rna isolation from cd + and (À) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd + cells express approximately fold tnfrsf and fold tnfsf when they are compared with control cells. tnfsf binds to cd that is expressed on the surface of tcells. cd does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf (dr ) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf and tnfrsf both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf and tnfrsf , these molecules may be essential markers to target cd + gbm stem cells. the effect of docetaxel on p , sin a and mdm gene expression in mcf- breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g /m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf- breast cancer cells line. in this study, mcf- breast cell line was applied different doses docetaxel ( nm, nm, lm, lm, lm) as h and h. mtt analysis was performed to the mcf- breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p , sin a and mdm gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < . ) in h as mtt analysis. the lowest cell viabilty was determinated in group applied lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < . ). the highest p , si a and mdm expressions were apperared in nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g ) and acetyl-terminated (g ) nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g and g dendrimer derivates labeled with dox were absorbed by the cells at °c with different efficiency. g -dox derivate characterized much slower internalization rate than nonacetylated g -dox. only g -dox shown partial colocalization with lysosomal marker lamp after h of incubation. internalization of afprbd-g -dox and afprbd-g -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp already after min of incubation. cytotoxicity results revealed that ic levels of g -dox and afprbd-g -dox coincided and demonstrated a bit higher activity against sensitive to dox skov and resistant skvlb cells than afprbd-g -dox conjugate after h of incubation. at the same time, after h of incubation afprbd-g -dox and afprbd-g -dox were much more than g -dox and g -dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. - - . ) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m -pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m -pk in breast cancer, and high m -pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~ . and . v potentials vs. ag/agci. m -pk activty was detected by using differential pulse method at between . and À . v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m -pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m -pk activity determination at low concentrations is possible with this method. p- . . - tie /tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie /tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie /tek in both cd + gscs and cd À gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with % fetal calf serum and % penicillin-streptomycin at °c in % co -humidified incubator. we isolated cd + and cd À cells from gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd À and cd + cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version . . tie /tek gene expression was determined as . fold higher in cd + gscs than normal brain tissue (p < . ). morever it was determined . fold higher compared to normal brain tissue in cd À (p < . ). according to our results tie /tek expression was higher in gscs, indicating that tie / tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no: s ). adenosine inhibited the breast cancer stemlike cell population through erk / pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk / protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk / pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd + /cd + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd + /cd + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for and h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd , cd , cd , cd and negative with hematopoietic markers cd , cd b, cd , cd and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for and h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in , high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the -phosphofructo- -kinase/fructose- , -bisphosphatase- (pfkfb ) regulates glycolysis by producing fructose , -bisphosphate (f , bp). given that pfkfb is induced by several established emt-inducers in tumor cells, e.g. hif- a and ras, we hypothesized that pfkfb may be involved in regulation of the emt in tumor cells. silencing of pfkfb in pancreatic adenocarcinoma cell lines panc and s vp was achieved using specific sirna molecules. mrna and protein expressions of the cdh gene (encoding e-cadherin, an established epithelial marker), as well as zeb and snai genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb was achived by transfecting cells with a plasmid carrying zeb cdna. cells that were depleted of pfkfb exhibited markedly increased cdh mrna and e-cadherin protein expressions and reduced snai and zeb mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb caused approximately % reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb depletion does not appear to be associated with reduced zeb expression, as ectopic expression of zeb did not reverse the effect of pfkfb silencing on invasion. taken together, these data suggest that pfkfb may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia hematology system and abbott cell dyn and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled . ae . x , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to . x , with a maximum value of x . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = . ), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia ( %), and chronic myeloid leukemia had % of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p- . . - effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type . the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst- tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at h time point. however, at h time point, hescs isolated with the non-enzymatic and collagenase type method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type isolation method at h, h and h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd and lower cd profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd , cd , and cd surface expression levels. only hescs isolated with collagenase type did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type , while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd + subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd . epcam+/cd + cells were sorted by magnetic separation from huh- cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd + subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd + cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd + subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su , in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd + subpopulation of huh- . in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd + hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg ) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst- assay and tg activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp- and - were investigated with zymography and rt-pcr, respectively. although tg activity was found to be % less in emscs isolated from endometriotic tissue, these cells showed times higher tg protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have . times higher tg and . fold higher itgb mrna levels when compared to the cells of healthy group. similar results were observed in sdc- gene expression with a . fold increase. endometriotic emscs demonstrated an average of . -fold increase in the mmp- activity while a onefold increase was evident in mmp- activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within h. the fact that emscs from the control tissue showed lower tg protein levels with a high enzyme activity suggested that tg might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg along with itgb and sdc was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp- and - . recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f with % fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd , cd , cd and cd (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd , cd for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd were decreased to - %. differentiation induced the expression of cd , cd for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~ % and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca + entry (soce) activated by depletion of intracellular ca + stores has been shown to control intracellular ca + homeostasis in many physiological and pathological events. stromal interactive protein, stim , as endoplasmic reticulum (er) ca + sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim and orai on soce in differentiation of huh- hccs expressing epcam and cd surface adhesion molecules (epcam + cd + ). epcam + cd + subpopulations in huh- cells were separated via flow cytometry and transfected with stim and orai- over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca + concentration were monitored via dual wavelength spectrofluorimeter in fura -loaded cells. in epcam + cd + cells, er ca + release increased without any change in soce compared to that of epcam À cd À cells. similar results were observed in stim -oe epcam + cd + cells. on the other hand, increase in orai has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost - % of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd , cd , nanog, oct / , hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd , cd , nanog and oct / significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf- release from human mesenchymal stem cells s. k€ ose , f. aerts kaya , d. uc ßkan c ß etinkaya , p. korkusuz stem cell research and application center, hacettepe university, ankara, turkey, department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor (sdf- /cxcl ) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf- release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage mscs were induced with ng/ml lipopolysaccaride (lps) for h. antagonists for cb (am ) and cb (am ) receptors were added to cultures for days. after incubation with antagonists msc culture supernatants collected and processed with human sdf- beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf- beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf- release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta- , igfr , vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n: ), media (m, n: ), stem cell in media (m+bmsc, n: ). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton ll saline, ll culture media and cell/ ll culture media. the pregnant female rats on the day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta- , anti-igfr , anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta- , igfr , vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol -kinase (pi k)/akt pathways. the effects of h s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h s agents on the in vitro cell growth and differentiation of human lin À sca + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h s-releasing agents, such as na s, gyy and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional d-systems for cardiac tissue repair, that take into account the relevant biological role of h s in the cardiovascular system. p- . . - investigation of the protective effect of boric acid and omega- fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into groups with rats in each: control, mi, mi+boric acid, mi+omega- and mi+boric acid+omega- groups. control rats were treated with ml/day saline, boric acid-treated rats received mg/kg/day boric acid and omega- -treated rats received mg/kg/day for days by oral gavage. for the experimental mi model, mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a -h interval in the last days of the boric acid and/or omega- treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp , biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < . , p < . and p < . respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < . and p < . , respectively) and pon levels were decreased (p < . ). according to the ecg recordings, st wave and heart rate were found to be decreased (p < . and p < . , respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega- after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega- after induction of mi. results of the present study suggest that omega- and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included patients diagnosed with dka, patients diagnosed with dk and voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values ( . ae . , . ae . . ae . ; p = . ). no statistically significant differences were found among groups with respect to ck-mb values ( . ae . , . ae . , . ae . ; p = . ). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group ( . ae . ; . ae . . ae . ; p = . ). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour after acidosis correction ( . ae . ; . ae . ; p = . ) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p- . . - genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac -hcms in dmem/f with %fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber ( %o , %co , %n ) for , , , h and the control group was incubated in normal conditions ( %co , %o ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u -plus- arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as h. we identified genes ( down-regulated and up-regulated) (p < . , fold change ≥ . ) were differentially expressed in hypoxic-ac vs. ac . degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif- signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number s . autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd and cd markers. purified ips cells were differentiated into flk + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days . to . . flk + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd and cd gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk expressing cell number reached to a peak level ( %) on day . followed by a progressive decline subsequently. in the second step, cd and cd positive cells were generated and enriched during day of induction. we showed optimal time for harvesting flk + cells is day . of initial differentiation. following isolation of flk + progenitor cells they were further matured into functional epcs by vegf within days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p- . . - electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups ( mg, mg, mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < . ). amplitude of p wave and qrs complex did not change (p > . ). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < . ). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t- rna polymerase, possess toxicity to leukemia cells hl- and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in - nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents ( - times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkÒ hydrogel photopolymerized in a d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd + stem cell subpopulation. cd + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkÒ hydrogel. printing was done using a lm diameter needle, under bar pressure, and mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd + cells viability is maintained after days of cultured in bioinkÒ hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd + cells expressed endothelial markers such as cd , cd , cd , cd and vegfr . in conclusion d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe o ) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe o were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at and days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe o functionalized with carvone and limonene significantly stimulated the production of il- , il- and il- , while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe o nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p- . . - new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between . and . mm and epinephrine concentrations between . and . mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be . and . mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has À . v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < . ). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than % for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, %); a positively charged phospholipid, dotap ( %) and cholesterol ( %) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll- is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll- . the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll- immobilized was determined by elisa method. . % of ll- peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll- immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using t mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll- immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio nanotube-modified ti surfaces (ti/tio ) on the cross-talk between raw . macrophages (mf) and mc t -e preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw . and mc t -e cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw . cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il- and mcp- release was significantly increased at h, while after days only il- exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for h and days, whereas the presence of the mf for weeks significantly inhibited mineralization. on ti/tio surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p- . . - synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan , , b. tural , s. tural mardin artuklu university, mardin, turkey, dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b and due to high collagen synthesis. on the other hand the other isoform tgf-b , suppresses the dense collagen production caused by tgf-b and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b was studied with elisa. hepg cell line was used on mtt cell proliferation assay for both tgf-b loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be . %. particle size, zeta index and polydispersity index for this formulation were determined as . ae . nm, . mw and . , respectively. thickness of the prepared films were ae . nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand s from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a -well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (% , % . , % wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (% , % . and % wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p- . . - evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz , s. dogan , s. celikler kasimogullari department of molecular biology and genetics, balikesir university, balikesir, turkey, department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations ( , . and %) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki , , c. dupont-gillain universit e catholique de louvain, louvain-la-neuve, belgium, j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of . kda, isoelectric point ip at ph . ) and lysozyme (lys, molecular weight of . kda, ip~ ) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly ( -(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph . (pdmaema is neutral in such conditions) and were swollen at ph . (pdmaema is positively charged). protein adsorption was performed at different ph values ( . - . ) and salt concentrations ( . - . m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [ ] , [ ] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, -amino pyridine and , -diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly( -hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe t (dsm ) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa ), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl- -pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu + -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p- . . - bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonÒ, we employed argon plasma treatment with the powers of and w for - s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst- reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p- . . - graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations ( . - wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from to days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a d composite hydrogel scaffold ( d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin À sca + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of d-chs for tissue regeneration using the emerging bioprinting technologies p- . . - gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformÒ, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite % and tricalcium phosphate %, with - % pore volume and - lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformÒ. in order to improve cell adherence, ceraformÒ was covered with fibronectin. the cells were cultivated for -day period by osteogenic induction medium. days , , , and were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor (runx ), bone morphogenetic protein- (bmp- ), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day th, continued to increase in day . bmp- gene expression was increased in , , day compared to day. on the other hand, runx gene expression was increased only on days and . these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p- . . - on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu , i. negut , f. sima , e. axente , l. e. sima national institute for lasers, plasma and radiation physics, magurele, romania, institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom ( . %) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to show negatively charged particles. when n/p was , the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than . the migration of polyplexes with different n/p ratios ( - ) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was %. tuesday september : - : aging p- . . - novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd - and vd - - ) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc values to be lm for vd - , lm for vd - - and lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc doses of vd - and vd - - showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd - - exhibited - nm ic for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited -fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd - - needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was -fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic of nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap- and on the activity of nfat and ap- transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß is more prone to aggregation to form plaques and more toxic to neurons than aß . in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß / in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin , namely ps , were treated with a dose range of tbo ( - lm) or vehicle control for h. after treatment, aß / levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß and aß secreted into the cell culture media. significant reduction for both aß species was evident at lm (p < . ), lm (p < . ), and lm (p < . ) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g and g cell cycle checkpoints by p and p mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > genes, including nine genes encoding transporters. p mapk and p were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p- . . - smad specific e ubiquitin protein ligase (smurf ) and its potential effects on inhibitory transmission in aging adams , , , interdisciplinary program in neuroscience, bilkent university, ankara, turkey, national nanotechnology research center (unam), bilkent university, ankara, turkey, department of molecular biology and genetics, bilkent university, ankara, turkey, molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, department of psychology, bilkent university, ankara, turkey smad specific e ubiquitin protein ligase (smurf ) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf , via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf transcript is significantly higher in old zebrafish brains. thus, smurf may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb /+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf and gep levels remain stable in the aging brain (p = . , p = . ), and in the ache mutants gep levels are increased compared to the wildtype controls (p = . ). further analysis of the relationships between smurf and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we evaluated patients with ihd and healthy subjects aged years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr rs and tlr rs were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr (arg gln) and tlr (asp gly) were not significantly different between the groups (p ≥ . ). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over years old (p ≥ . ). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we plan to include other types of polymorphisms in tlr and tlr genes and increase the volume of patient cohort in our future studies. p- . . - evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up % and hemorrhagic strokes % of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke patients, with acute ischemic stroke and healthy controls we reenrolled in the study. blood samples were taken within st and th days, and after rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: . ) and thiol (p: . ) levels were significantly higher in first h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the rd month values of tos and nihss score (r = . , p = . ). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p- . . - amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from infertile women with pcos and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p promoter of xp bacteriophage in presence of p protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp , in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p protein, except of bacteriophage p promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p protein in the transcription initiation and identification of the role of the individual elements of p promoter of xp bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p protein. we have designed a set of promoter probes representing the combination of sequences of p -resistant p promoter and p sensitive t n promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream À position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing À promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region , a sigma-factor domain responsible for interaction with À promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p protein. promoter probe with up-element of p promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p promoter of bacteriophage xp can escape inhibition by p protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p- . . - distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study gerbils brains were used and divided into groups (n = ): : newborn animals ( day), - : , and days respectively, : animals aged years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within days (in cerebellumamounted to . ae . lg/ mg tissue; to - days increased to . ae . lg/ mg tissue, and began to grow again in older individuals aged years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at days p.d. ( . - . lg/ mg tissue), and unchanged for days. these results revealed that the most intensive development of astrocytes in the cerebellum to p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs . in order to investigate the impact of whirly on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly (hvwhy kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs at onset of drought-induced senescence in wild type (wt) and hvwhy kd lines, stress-responsive loading with histone modifications at gene regions of hvs ( regions in the promoter, one region around translation start site and regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy kd indicating that whirly protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h k ac all over the analyzed gene regions, correlating with a massive induction of hvs expression, while drought stress caused no substantial increase of h k ac in why kd plants. the results suggest that drought induced expression of hvs is under epigenetic control, and furthermore that why is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after week of inoculated with erlich cancernome ( cells/animal, i. p.) in months male balb/c mice treated at once with or without plant extract with bbv ( mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on % and % respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold ( . % versus . % in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at lg/ml concentration against escherichia coli vkpm-m , pseudomonas aeruginosa grp , bacillus subtilis wt-a , salmonella typhimurium mdc and staphylococcus aureus mdc , candida albicans wt- and candida guilliermondii hp- . statistical analysis was done using graphpad prism . . crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p- . . - effects of aluminum stress on photosystem-i apoprotein a gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl in different doses ( . , . , and mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ . ) decreased after hours treatment with mm and mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with . and . mm al for hours increased the gene expression level of psab by . % and . % respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, mm al exposure for hours could damage the electron transport in photosystem i. p- . . - nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + mg/kg ns; iii = sham + mg/kg (n-acetyl cysteine) nac; iv = g/ kg paracetamol; v = g/kg paracetamol + mg/kg nac; vi, vii and viii = g/kg paracetamol + , and mg/kg ns, respectively. paracetamol administration (oral) was carried out h after ns and nac administrations (oral), and all animals were sacrificed h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p- . . - the study of problems of preservation of the birches e. shadenova , , e. zhumabekov , m. sembekov , m. burchaeva institute of general genetic and cytology, almaty, laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p- . . - fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht- , t and upci-scc- cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a ( . m/z) and -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at th and th hours compared to control group. this study shows that holothurin a, dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support ( z ). p- . . - alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified genes encoding sr proteins in chinese cabbage (brassica rapa chiifu- ) from brassica database and analyzed their phylogenetic relationship. b. rapa has types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage ( and days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra and bra were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p- . . - characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of u mg(- ) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to . . the optimum ph and temperature was estimated as . and °c, respectively. the purified enzyme was stable from ph . - . and able to thermal stability up to °c. the k(m) and v(max) values were . mm and . micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf- , substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd + and hg +. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, , , -trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in , but the uniprot database contains less than reviewed sequences, which is approximately -fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above , are easily detectable in the total protein extracts. p- . . - transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in transcripts, database search with trinotate tool revealed transcripts with aox domains characterized in known alternative oxidases. blast analysis of these sequences with known aox proteins revealed three distinct aox genes (nalba-aox , nalba-aox and nalba-aox ). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox , nalba-aox are predicted as mitochondrial while nalba-aox is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr rk ). p- . . - comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak , v. koukalov a , z. medvedov a , c. martin , j. hradilov a , l. sp ıchal , b. brzobohat y mendel university in brno, brno, palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde ( -chloro- -( -methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of -day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p- . . - the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as °c and , respectively. the enzyme was stable at °c, but thermal stability was dramatically decreased at higher temperatures up to °c. kinetic parameters were also measured; vmax was . u/ml min and km was . mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp , lira and lines g- / _o, g- / _k were treated for and hours with lm alcl solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about million raw reads were obtained and conserved mirnas from families were identified. significant expression alterations in flax plants under al treatment were shown for mir and mir . expression level of mir was varied in similar way in resistant and susceptible to al genotypes: mir was up-regulated after hours of alcl exposure and down-regulated after hours. mir expression was increased after hours of alcl exposure and decreased after hours in susceptible to al flax genotypes (lira, g- / _o), while in resistant genotypes (tmp , g- / _k) mir level was decreased after both and hours of al treatment. in other plant species, mir and mir were identified as al-responsive. mir targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir targets mrna of tas protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir and mir in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir in susceptible and resistant to al genotypes. this work was financially supported by grant - - from the russian science foundation. p- . . - association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from year old poplar trees ( clones x replicates x ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as . ae . lg/ml, ae . lg/ml, and ae . lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p- . . - proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum ( min), radio-frequency ef ( - min) and cp ( - min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in days in comparison to days after seed treatment. the d dige analysis revealed differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin- were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p- . . - suppression of lipopolysaccharide-induced inflammatory responses in raw . macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw . macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il- concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw. . cell were found as . - lg/ml. studied cyclamen taxa inhibited tnf-a and il- release on lps stimulated-raw. . in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p- . . - in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < . ). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < . ). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p- . . - cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase- expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp- mrna expression of human sw colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp- mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag- s . data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with . lm ic value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at lm concentration when compared to control and lm (p < . ). cub alone showed inhibitory effects on mmp- mrna expression at lm and higher doses significantly (p < . ). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp- expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp- synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using % aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between mg/ml and . mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to % sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc is gram-positive cocci (mic: mg/ml). the mic values of other gram-positive bacteria s. aureus atcc , e. faecalis atcc , l. monocytogenes f and m. smegmatis cmm were detected as , , and mg/ml respectively. mic values of gram-negative bacilli were detected as > mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc , s. aureus atcc and e. faecalis atcc ). the strongest effect was observed on s. aureus atcc strain. in contrast, extract showed less effect on gram-negative bacilli. p- . . - selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a ) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p- . . - dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu , f. zihnioglu denizli vocational school of technical sciences, pamukkale university, denizli, department of biochemistry, faculty of science, aegean university, izmir, turkey type diabetes mellitus (t dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide- (glp- ) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec . . . ) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic values were also determined by nonlinear regression curve fit using graph pad prism . with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic . lg/ml while the standard (positive control) diprotin a displayed the ic value of . lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p- . . - dna recognition determinants of arabidopsis thaliana b transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b tfs constitute a large, plant-specific protein family (approx. % of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. amino acids) b dna binding domains. currently the b tfs are divided into four groups (lec -abi /val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b domain (arf , arf family) is available, thus the mechanism of site-specific dna recognition by the lec -abi /val and rav b domains remains poorly understood. based on the arf -dna structure (pdb ldx) we have built homology models of dna-bound b domains from a. thaliana abi (lec -abi /val family) and nga (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi / nga b domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b domains and in the future may help us predict the dna binding properties of currently uncharacterized b domains. p- . . - immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili , a. zeytunluoglu denizli health services vocational high school, pamukkale university, denizli, denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp- and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group : control, group : stzinduced diabetic, group : o. hypericifolium injected, group : stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups and by a single intraperitoneal injection of stz at a dose of mg/kg body weight. in groups and , rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of ml/kg body weight for consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine (k ) of tubulin. k is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk , e. marusich , r. chuprov-netochin , j. neskorodov , y. mishutkina , s. leonov life sciences center, moscow institute of physics and technology, dolgoprudny, center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature ( °c) and light conditions ( : hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point- . od) and hours later. to validate our system we used known cytokinins and auxins ( mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in - times range, compared with only times increase in control group. eight new chemicals ( lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to %. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an % yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl , pl , pl , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total sequence tagged site (sts) sequences from ncbi were aligned for pl , pl , pl , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p- . . - investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg cells line s. altundag , pamukkale university, denizli, istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg - cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut- ,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol Ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut- ,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut- ,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg - cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut- ) analyzed the expression levels . teucrum polium plant extract is applied in hepg - cells the glycolysis pathway genes (pk m glut- ) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg - cells showed antidiabetic effect. p- . . - purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara , s. sinan , z. ekmekci , y. turan university of balikesir faculty of veterinary, balikesir, university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec . . ) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with % ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose b-ltyrosine- -naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph - and thermal optima was determined using - °c temperature range. inhibitory effects was determined with mm substances. results: the enzyme was . -fold purified with yield of %. purified b-glucosidase from apricot seed was visualized about kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of . mm and . eu, respectively. the optimum ph and temperature were determined . and °c respectively. effects of cacl , kcl, nacl, mgcl , k so , na so , cuso , fecl , pb(ii) acetate, agno , zncl and glucose on purified enzyme activity were investigated. kcl, na so , pb(ii) acetat and cuso reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p- . . - a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un , , o. arslan balikesir university, science and technology application and research center, balikesir, department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec . . . ), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose- b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose- b-aniline p-amino benzoic acid affinity chromatography gel. % . yield and . fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of kda. the v max and k m of the purified enzyme were determined , u/ml min and . mm, respectively. p- . . - phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken , b. kocat€ urk , s. dogan , h. tuner balikesir university, balikesir, kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have , , kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of s promotor region in fragmented dnas was proved by pcr. escherichia coli dh a strain was transformed by fragmented dna samples according to cacl method. s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to - bp for bacterial transformation. fragmented dnas confirmed for carrying intact s promotor sequence. none of bacterial genomic dnas were found to be s-positive. according to the transformation results, absence of s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p- . . - preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker (tm- ) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p- . . - cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in , minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima - , tm- and ps- were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in % high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a °c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p- . . - association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of - nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm- (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p- . . - pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of chls, a homozygous cotton line tm- and a double haploid line pima - were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm- and pima - (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p- . . - pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during - . chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy ( n = - ), polyploidy ( n = - ) and the occurrence of additional b-chromosomes ( n = + - b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of s rdna and s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to . %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p- . . - angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu , i. arslan biomedical equipment technology, pamukkale university, denizli, turkey, denizli, biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the kda < peptides < kda in the ultrafiltration fractions displayed highest ace inhibition ( . ae . % at lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p- . . - modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg cell line d. irtem kartal , a. altay yuzuncuyil university, van, erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than different ailments ranging from aches to epilepsy. there are around species of salvia, of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm and catalase in hepg cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg cells for hours and hours incubation and methanol extract decrease catalase mrna expression for only hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing gigabases of bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcÀesiÀqtofÀms) to identify drought-related proteins. totally proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p- . . - isolation and characterization of a hexose converter from olive s. altunok , e. d€ undar department of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer , finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was kda with a pi of . . the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number o . key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p- . . - antioxidant potentials of propolis and its bioactive components, and their effects on cyp e gene expression in ht- adenocarcinoma cell line a. altay , d. irtem kartal erzincan € university, erzincan, y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p enzymes are involved in phase i xenobiotic and drug metabolism. cyp e is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with % ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht- human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp e in ht- cells were investigated bu using q-rt-pcr technique. ic values for dpph radicals scavenging activities of the extract was calculated as . mg/ml. tpc and tfc were determined as . mg gae/g extract and . mg qe/g extract, respectively. caffeic acid content of the extract was measured as . lg/g extract. ic values for xtt assay in hours incubation with the extract and caffeic acid were evaluated as . mg/ ml and . mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp e mrna expression with . and . fold, respectively in ht- human colorectal adenocarcinoma cells for hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp e expression and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - effects of histone h lysine inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h at lysine residue by specific methyltransferases suv h in humans and kyp/suvh in arabidopsis generates a'code' for the recruitment of hp proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv h has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h k methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf- breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt ( , -bis( -metoksi- -nitro- -sulfofenil)- h-tetrazolyum) test. in order to determine the dose of ic , plant extracts were applied as time and dose dependent in the range of - ug. in nd hour, the ic value is determined as ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase- , caspase- , caspase- , caspase- , bax, bcl- , fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while . and . -fold respectively increase in the gene expressions of caspase- and caspase- of dose group cells, . -fold decrease in the gene expressions of bcl- . no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p- . . - the effect of ferulic acid and -fluorouracil combination on apoptosis in pc- human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. -fluorouracil ( -fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; -hydroxy- -methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and -fu on apoptosis in pc- human prostate cancer cell line. the effects of -fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp , casp , casp , casp , bcl , bax, fas and cycs were investigated in four groups by qpcr. the ic doses of fa and -fu were found to be lm and lm for hours in pc- cells, respectively. in order to determine combination effect, pc- cells were treated with <ic doses ( lm fa and lm -fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl were observed in the fa group, compared with the control group cells. after the treatment with -fu, the expression of casp was significantly increased compared with the control group. furthermore, combination of fa and -fu significantly increased expression of casp , casp , fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and -fu affected expression of different genes in apoptosis in pc- cell line. p- . . - combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc- human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc- human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp , casp , casp , casp , bcl , bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic doses of fa and gemcitabine were found to be lm and lm for hours in pc- cells, respectively. for determination of combination effect, pc- cells were treated with <ic doses ( lm fa and lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp , casp , casp , fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc- human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf- , hela, a , and ht- cancer cell lines and nih- t cells. the viability of cells in lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic = . ae . mg/ml) and abts radical cation scavenging activity ( . ae . mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc ( . ae . mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species ( - mm / < . - mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain ( - mm/> mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p- . . - antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention ( ) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms ( ) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: ), aureus e. coli (atcc: ), and salmonella enterica (atcc: ) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with and milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be milligrams and milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with and milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p- . . - molecular analysis of serine/arginine rich sc splicing factor from olive b. bas , e. d€ undar depertmant of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc splicing factor (oesarsc sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc sf was a protein consisting of amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was kda with an isoelectric point (pi) of . . the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number o . keywords: olea europaea l., oesarsc sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p- . . - application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli , g. arabaci kto karatay university faculty of medicine, konya, faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h o and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with % (w/v) ammonium sulfate ((nh ) so ) and added t-butanol with the ratio of : . (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh ) so ( , , , , %) and tbutanol ratios ( : . , : , : . , : ) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were . , . % and . , . . therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of : . , the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh ) so ( , , , %) were . , . , . , % and . , . , . , . . the interfacial phase was not dissolved in % (nh ) so . at constant % (nh ) so , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio ( : . , : , : . , : ) were . , . , . , . % and . , . , . , . . finally, at optimum conditions (% (nh ) so , t-butanol : . ) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were . % and . . the results of gel electrophoresis showed that the molecular weight of enzyme was between - kda. the applications of tpp gave the maximum recovery of . % and . -fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for , and days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of distinct spots in brachypodium protein repertoire. a total of differentially expressed proteins (deps), with at least -fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p- . . - immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m e epitope from matrix m protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m e sequence. accumulation level of m e-ala recombinant coat protein was significantly higher than m e-cys/ ser (ratio : ). tmv-m e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves ( d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm ae . cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw . macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase- (cox- ), tumor necrosis factor-a (tnf-a) and interleukin- (il- ) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox- and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il- in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p- . . - the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf- breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf- breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase , bax and p and antiapoptotic genes; bcl- and pi expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf- cell was only observed in and lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf- cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf- breast cancer cell lines. p- . . - reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are , bp (mon- kalr) and , bp (mon- volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and ribosomal rna genes. and trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf , ycf , accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant - - and rfbr grant - - (mon- volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), -methyl-z-tetradecen- -ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, -octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p- . . - role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut , deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature ( k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p- . . - the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature ( / °c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor , -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for days at optimal conditions. d was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p- . . - reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m ) was investigated. three weeks old tomato grown at optimal conditions ( / °c day/ night temperature and light intensity lmol/m /s) plants were exposed for and days to elevated temperature at / °c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf- cells. mcf- cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively , and ng/ml) . then different doses of silymarin ( , , , lm), curcumin ( , , , lm) and propolis ( . , . , . , . mg/ml) were added. after , , and hours incubation periods different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf- cells for , , and hours (biovision cell proliferation kit) all experiments were carried out different days, at least times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < . between and hours. it was found that there were not seen much cells at and hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was . mg/ml, silymarin and curcumin were lm. also, we evaluated the effects of on proliferation rate the mcf- cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf- cells in hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p- . . - investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; -hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, h-nmr and melting point analyses. the theoretical uv-vis spectra and d conformations of the isolated compounds were determined by using the gaussian software with hf theory at the b lyp/ - g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [ , ] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than hydroxyphysodic acid, and the toxic activities were found sufficient as much as % at the highest doses of the substances; lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately % by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [ ] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. ( ) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research : - . [ ] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. ( ) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian software with hf theory at the b lyp/ - g level was also performed to determine the theoretical uv-vis spectrum and d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [ , ] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as % at the highest dose of the substance; lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as , and -lm, and % of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size ( - nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n is %, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score or higher, p < . ) peptide mass fingerprint of cwr -de electrophoresis revealed differences in protein composition between cwr and n . information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p- . . - mir and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir s and their target by q-rt-pcr. we mined mir genes sharing sequence similarity and grf targets. the expression analyses of mir s and grf are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir s and grf in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate , and to detect expression through real-time pcr were designed using primer . amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as . . the molecular weight of the protein was calculated as kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p- . . - cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht- , t and upci-scc- cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for hours. the cell index data were compared with the control group. ic values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a ( . m/z), -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). the fraction d was the most effective on all cell lines with ic value of . ae . mg/l, . ae . mg/l and . ae . mg/l for ht- , upci-scc- and t , respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p- . . - effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol ( %) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose ( mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically % (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp- , mmp- by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of rd, th, and th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the rd and th days. the malondialdehyde levels were significantly lower in the treatment group. mmp gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the th day while it was reduced in th day. mmp gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in th, and th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp and mmp regulation. p- . . - isolation and molecular molecular characterization of vps /vam from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps /vam promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps /vam , however, have been reported. the aim of this study is to report information of olive vps /vam including expression pattern and biochemical characterization. vps /vam was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega . cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn d. the blast and bioedit analyses revealed vps /vam had base pairs coding amino acids with a molecular weight of . kda, and pi of . . the at/gc ratio was very high ( . ) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the d structure of the protein was similar to its previously reported homologs. p- . . - isolation and characterization of the ribosomal l protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l protein is larger than the lsu of ribosome and contains domains as -layered alpha/beta domain and -layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l (rl ) gene has been studied in many plants, reports on olive rl (oerl ) are very rare. this study presents molecular characterization of rl gene from olive. oerl was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional ( d) structure analysis, and phylogenetic analysis were conducted using signalp . , thhmm, i-tasser/cn d and mega , respectively. oel was found to have an open reading frame of base pairs coding amino acids that constitutes a molecular weight of . kda and a high pi of . . lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a . at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl . likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl with respect to expression pattern and biochemical function continues. p- . . - isolation and characterization of an olive splicing factor b subunit m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor b subunit (sf b ) functions in the regulation of translation and gene expression. sf b forms u small nuclear ribonucleoprotein complex (u snrnp). splicing factors a and b binds pre-mrna at the site of the intron branching point. this binding joins u snrnp to pre-mrna. although sf b from various plants have been widely studied, no studies on olive sf b (oesf b ) have been reported. this study reports information on various aspects of oesf b . oesf b was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn d were used to generate the calculated d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb . the orf contained nucleotides coding amino acids that produce a . kda peptide with a pi of . . alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf b is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf b could be synthesizing in mitochondria. the predicted d structure of oesf b was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p- . . - kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of - %. they are grouped into a ( - kda), b ( kda), c ( kda) and d ( kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of % naci concentration, and second, kafirins were obtained using tertiary butanol ( %) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as , , , and %. of the total proteins, % was identified as a, % b, % c, % d, and % non-prolamines. non-prolamin group of proteins were visualised as different bands ranging from . to kda. c and b group of proteins were only viewed when treated with reducing agents as -me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for . , . , . , . and . kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p- . . - untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu , university of agricultural sciences and veterinary medicine, cluj-napoca, center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol ( ) and chloroform/methanol ( ) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction ( ) , ranging from . to . mg/ g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. / , pncdi . glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys ko) and liver glycogen synthase (gys ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos , nos (genes encoded no producers) and pde a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos which lost rhythm and increased and delayed amplitude of nos expression. further, pinealectomy initiated cyclic transcription of gucy b and noncyclic transcription of gucy a (genes encoded cgmp producers) and increased mesor and amplitude of pde transcription. the transcription of prkg , the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi or other investigated genes (coxi , nrf , nrf a, pgc a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s fraction mix was used. cells were treated with . , and lg/ml oseltamivir, the tamiflu capsule ingradient, for or hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ . ) in the absence of s mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: . - mm of glucose, . - mm of urea, and . - mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p- . . - ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide ( amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with mm dimensions. ovary adenocarcinoma cells (efo- and onco-dg- ) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak- s ). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide ( n) contains biotin on '-and '-ends, and sterptavidin in molar ratio : . this complex represents a structure with equivalent electrophoretic mobility of bp dna and preferred "valency" of streptavidin is . this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved - times highter sensitivity than elisa. to reduce the matrix effect we used - fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only mkl of serum sample). antibodies to the disaccharide galb - glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ . ) for health donor sera (n = ; . ae . pg/ml) compared with sera from patients with breast cancer (n = ; . ae . pg/ml). sensitivity of igm anti-le c antibodies detection was pg/sample ( mkl) ie . molecules. thus for the immuno-pcr detection of antibodies the - tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (# - - ) and by russian federation president scholarships donated to d. yu. riazantsev (# sp . . bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma , as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = , the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge ( uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = , when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle- cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, u of penicillinstreptomycin and maintained in a humidified incubator at °c and a % co atmosphere. cell viability was measured using the colorimetric -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in well plates and incubated with ll/ml dmso, - lg/ml tm, . - mm tudca, or lg/ml tm + . - mm tudca for - hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for hours at °c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or mm tudca was cytotoxic. at and hours incubations mm tudca and lg/ml tm + mm tudca were significantly cytotoxic compared to control, dmso and mm tudca groups. treatment of cells with . mm tudca hours before administrating ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at mm concentration when treated with tm. therefore . mm of tudca, administered hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; s ). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a (lp-pla ) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla mass, activity, index) related genetic variations (pla g ) increase the risk for developing future cardiovascular disease in women with pe. a group of preeclamptic patients and normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla mass level. lp-pla activity were determined by kinetic method. plag snp genotyping performed by using the sequenom massarray iplex. the rs tt genotype had a higher lp-pla index (p = . ) for early onset preeclampsia, cc genotype had a higher lp-pla mass and lp-pla index for late onset preeclampsia. no difference were found for control. the rs gg genotype had higher lp-pla mass and index for late onset preeclampsia (p = . , p = . respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs and rs snps in early, late onset preeclampsia and control group. only lp-pla mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla mass, index and rs , rs snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi- mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding -hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi- mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found ae , ae , and ae hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi- mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi- mutation. it could accurately discriminate between normal and ivsi- mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il- b and il- and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in and hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio . to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il- b, il- and tnf-a genes were increased by the exposure to nanoparticles of , and lg/ml for - hours. tio nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio . oxidative stress was elevated by the treatment with tio nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il- b, il- . background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor- (vegfr ) and a v b -integrin on d and d cultures (spheroids) of ea.hy . methods: the ea.hy cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf ( ng/ml) or vegf a ( ng/ml) for hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr and a v b -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b -integrin and vegfr- increased on the d culture compared to d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b -integrin in the d culture of the ea.hy cell line activated with tnf. also increased expression of vegfr in the d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b -integrin and vegfr on the surface of d and d cultures as well as antibodies with fluorescent label. conclusions: d cultures of the ea.hy cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the d structure of tnf and ii) design of more potent and less toxic spd analogues. b. drug synthesis. a series of spd analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded %. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd derivatives have better dissociation constants than spd . the effect of exercises on serum bmp- levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp- is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp- to sustain the molecular approaches for treatments. a total of patients were included in this study. the groups are formed as follows: group , oa patients before the exercise; group , oa patients after the exercise; group , oa patients before the isokinetic exercise; group , oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after weeks of the exercise programme. self reported severity of pain was measured using the mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp- levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp- levels were significantly decreased in group compared to group ( . ae . ; . ae . respectively, p < . ) and in group compared to group ( . ae . ; . ae . respectively, p < . ). there is not any statistically differences between group and group (p > . ). as a conclusion, the decreased serum levels of bmp- may be suggested as a biochemical marker for oa patients during exercise programmes. p- . . - tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase- ), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < . ). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase- ) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < . ). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < . ). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p- . . - short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin ( mg/kg, i.p.). rats were divided into group i: control, ii: control/ mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/ mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il- b, il- (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il- b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p- . . - cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb- and mcf- breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb- and mcf- breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb- wt and mda-mb- gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as lm (for mcf- ) and lm (for mda-mb- ) decreased cell proliferation and induced apoptosis through causing jak dephoshorylation, stat , , dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for hours. lm curcumin-induced apoptotic cell death via dephosphorylating jak at tyr / residues and decreased phospho-stat , level in both breast cancer cell lines. although curcumin dephosphorylated stat in both mda-mb- and mcf- wt cells, no significant effect has been observed in mda-mb- gh+ and mcf- gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf- and mda-mb- breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat , a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb- breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb- breast cancer cell lines. we found that lm curcumin-decreased in apoptotic cell death through inactivity at jak which led to dimerization of stat , stat , stat . concomitantly, curcumin affected stat regulating socs proteins in mda-mb- breast cancer cell line. in addition, we demonstrated that lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb- breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il and il cytokines as main mediators of inflammation progression, correlated in keratinocytes with il a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam- and vcam- adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il and il in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam- over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into groups (n = ). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + mg/kg salvia l.group; group iv: i/r + mg/kg salvia l. group; group v: i/r + mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for days was given single dose as a gavage. minutes ischemia, minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)- and interleukin ib (il- b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il- and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il- , and il- b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group mg/kg salvia l. and mg/kg rosmarinic acid especially mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but mg/kg salvia l. extract were more effective than mg/kg salvia l. extract and used as synthetic mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a (spla ) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla , lpl and evaluate changes in circulating protein levels of angiopoietin-like protein (angptl ), angptl , cyclooxygenase (cox) and prostaglandin e (pge ). serum from control subjects and acne vulgaris patients with moderate and severe disease was evaluated for levels of spla , cox, pge , lpl, angptl and angptl . disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au clinical chemistry and unicel dxi immunoassay systems). serum levels of spla and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge , angptl and angptl levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla , lpl, angptl and angptl levels in acne vulgaris. future studies are aimed to understand the regulation of spla and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; # s ). p- . . - -ohdg and hogg levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. -hydroxy- -deoxyguanosine ( -ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human -oxoguanine dna glycosylase (hogg ) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after months of isotretinoin treatment by measuring -ohdg and hogg levels. the current study includes acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. -ohdg and hogg levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg and -ohdg, respectively.both -ohdg (p as a conclusion, isotretinoin increases dna damage and high serum -ohdg and hogg levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor (spi- ) in the stat -mediated manner in pinealocytes. however, the precise function of stat in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi- in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi- were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat transfection. the successful transfection and subsequent decline in stat expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi- were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat on those immunomodulators is important from the clinical point of view because inhibitors of stat are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles ( nm) using human lung mrc- fibroblast as model. cells were exposed to . lg/ml silica nanoparticles for , and hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il- b, il- , il- , tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp- , mmp- , mmp- ) and tissue inhibitor of metalloproteinase- (timp- ) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il- , il- , il- and tnf-a as well as of mmp- and timp- , was up-regulated whereas those of mmp- , mmp- was down-regulated after and hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase- in mrc-cells after and hours. p- . . - association of fto gene variant (rs ) with markers of t dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type diabetes (t d) encodes a -oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included patients with t d and prediabetes and healthy controls both sexes, aged from up to years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs with increased risk of t d (or = . , % ci . - . , p = . ). a allele was significantly associated with higher values of hba c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb ), glial fibrillary acidic protein (gfap), c-x-c motif chemokine and (cxcl , cxcl ), matrix metalloproteinase (mmp ), chemokine (c-c) motif (ccl ) and tyrosine-protein phosphatase non-receptor type (ptpn ) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak s and pamukkale university paubap fbe . p- . . - apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek , s. savranoglu , a. r. t€ ufekc ßi , f. g€ ul , i. demirtas , t. boyunegmez t€ umer graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw . macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into subfractions by column chromatography. e was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw . cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e , and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at lm decreased no production and %-ic : and lm-by inhibiting inos gene expression and % as well as protein expression and %, respectively (p < . ). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id: t ) support this work. the role of tip in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il- (interleukin ) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip conditional knockout mice, so we thought that tip may have a direct or indirect role on inflammation mechanism. tip (tat interacting protein, kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il- were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il- was increased in lps-induced inflammation group (p < . , p < . ) and liver-specific tip conditional knockout group mouse liver (p < . , p < . ). our initial data suggest that tip may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: z ). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose -phosphate dehydrogenase (g pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il ), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il was significantly increased in lps treated group. while the level of mda and h o was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: z ). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < . was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = . ) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of patients which have been diagnosed with cchf. matched for gender, healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p- . . - inhibitors of mdm ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm is an e ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p / mdm interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm and p might be playing a substantial role in autoimmune processes. we used a small molecule p /mdm inhibitor nutlin- a to test the effect of p activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p /mdm interaction by nutlin- a alters the p levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p /mdm interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant - - mol_a_dk. can ykl- be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc . and netphos . program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into groups: deletion/deletion (dd) genotype (group , n = ), insertion/deletion (id), insertion/insertion (ii) genotypes (group , n = ). blood pressure and pulse measurements were performed in all patients within minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes ( . ae . and, . ae . , p < . ). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone ( mg/kg b.w.) with and without iu of d (for days). the levels of rank, rankl, opg, a-hydroxylase (cyp b ) in bm were determined by western blotting. vitamin d receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. ohd content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp b , which is responsible for , (oh) d synthesis, in bm and ohd content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d and rank/ rankl/opg pathway. vitamin d co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d treatment. p- . . - heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor encoded by the hsf gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa a promoter (hsp promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp inhibitor, mg as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks and tks , the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks binding partners including adaptor proteins itsn , itsn , crk and grb , kinase src, amph , bin , plcg and also another member of the tks family -tks . it may indicate the possible role of tks in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph and bin , as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks isoform -tks -beta. we suggested that an active state of tks is regulated via intramolecular interactions between its proline-rich motifs and own sh -domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr and wire interact with sh domains of itsns in complex with actin. p- . . - correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in % methanol. after sample preparation obtained solutions were analyzed by hplc-agilent with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp within insulators still remains to be elucidated. recently we described an interaction between cp btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate aa fragment within ctcf c-terminal domain sufficient for interaction with cp btb, but deletions of flanking regions also lead to the loss of interaction with cp in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp insulator protein at . a resolution. overall structure is similar to other btb-domains. cp btb-domain has peptide-binding groove similar to that previously found in bcl btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project № - - ). p- . . - comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f hybrids of romanov sheep with . % of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by . % lower ( . ae . %, p < . ), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by . % and . % higher ( . ae . and . ae . (p < . ), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from . % to . %) and for oleic, linoleic, arachidonic acids (increased from . %, . %, . % to . %, . %, . %, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was . ae . , . ae . and . ae . %, that was . % lower (p < . ), and . % and . % higher (p < . ) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. - - . foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd (nbd ) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature > °c). the genome of nbd is $ kb long, and has a total of probable protein-encoding genes as well as gene for trna ser . the genome analysis revealed that nbd orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, are similar to proteins from phages that infect different members of enterobacteriaceae, while nbd orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, nbd orfs were given a putative functional annotation, including genes coding for morphogenesis-related proteins, as well as associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit- / ) from the research council of lithuania. p- . . - the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas , j. williamson , v. schw€ ammle , s. douthwaite universidad de cundinamarca, cundinamarca, colombia, university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as , as -rlma::aph and b) were grown aerobically in nutrient rich lb media at °c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be > -fold higher the wild-type b-strain of e. coli than for the hypersusceptible as strains ( lg/ml). whole genome comparison of the b and as strains were characterized by an increase in proteasome components ( proteins), chaperones ( ), error-prone dna polymerase components ( ), ribosomal hibernation factors ( ), heat shock response ( ), electron transport coupled proton transport ( ), pentose phosphate pathway ( ), flagellar assembly ( ), oxidative phosphorylation ( ) and tca cycle ( ). whereas ribosomal proteins ( ), aminoacyl-trna synthetases ( ), rnases ( ), abc transporters ( ), mismatch repair ( ) and sec secretion pathway ( ) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak /stat signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e g), asparagine to aspartic acid (n d), threonine to alanin (t- a) missense mutations, alanine to serine (a s) substitution, tryptophan to stop codon (w- x), gaaa insertion in intron of gh-n gene and both intron (+ c) and deletion of . amino acid of gh protein phenylalanine (f del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc . vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w- x, e g, f del, a s and n d mutations suppresses gh signaling via acting on either jak dephosphorylation or stat downregulation, t- a, gaaa insertion and deletion of + c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek cell lines. as a result, w- x, e g, f del, a s and n d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no tyr)-containing proteins were immunoprecipitated from serum with a no tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no tyr antibody. among the numerous immunoreactive bands observed in disease patients, the kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha- (iii) chain (co a _hu-man) with mascot algorithm analysis giving a score of (p < . ). collagen alpha- (iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no tyr modifications in co a relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs in gckr gene and rs in galnt gene with parameters of t d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved patients with t d and healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt polymorphism (rs ), while polymorphism in gckr (rs ) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs variant with waist circumference (p = . ) and fasting glucose levels (p = . ) in the control group. no such association was demonstrated for rs galnt gene. in the group of diabetic patients, significant association of gckr rs variant with levels of bilirubin (p = . ) and rs galnt variant with hba c (p = . ) and triglyceride levels (p = . ) was also demonstrated. our results suggest an association of variations of gckr and galnt genes with specific markers of t d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp binding site of the collagen type a (col a ) gene is associated with low bmd. we examined the distribution of col a gene polymorphism in young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤ . sd and controls was healthy women ( - years). mean age ( . ae . ) and ( . ae . ) respectively. the bmd, as g/cm , was measured in the hip and the lumbar spine (l -l ) with (dexa). dna was isolated from blood. col a gene was analysed with genomica clinical array system. the x test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was À . ae . . mean bmd (as g/cm ) was . ae . , and ( . ae . ) genotype distribution were ( %) ss, (% )ss, (% )ss for patients, and ( )ss, ( )ss, ( )ss for control . patients had (% )s allele, ( %) s allele, controls had ( %)s allele, ( %)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > . . genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(% ) than controls (ss % ) p < . . this study shows that high prevalences of the ss genotype at the col a locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col a and col a mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col a gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly ( - %) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep b at - and hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep b cells in three different concentration, namely - and . lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was lm. proapoptotic genes, bax and bad, and autophagy genes, beclin- and lc , were upregulated in hep b cells after hours treatment in contrast, antiapoptotic gene, bcl- , expression level was decreased for hep b cells after hours. p- . . - association of irs genetic variation with type diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate- (irs ) encodes the irs protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs single nucleotide polymorphisms (snps) were associated with type diabetes mellitus (t d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs (g>a), rs (t>c) and rs (a>t) with t d risk and related traits. our study involved t d patients and healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss . our results demonstrated a significant difference in frequency of rs (p < . ) and rs (p = . ) snps between t d patients and control subjects. interestingly, here we showed a significant association of irs rs risk t allele with increased insulin levels (p < . ) and homa-ir (p < . ) in t d patients. similarly, rs variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = . ) and homa-ir (p = . ). no such association was demonstrated for rs . however, this irs variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = . ) and decreased hdl levels. our results suggest that irs variants are associated with t d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t d biomarkers. the adra a gene encodes alpha- a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra a polymorphisms (rs a>g and rs g>t) with type diabetes (t d) and its related traits. in this study we have included t d patients and healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics software. our data showed that frequencies of both, rs and rs , variants were not significantly different between t d and control subjects. however, rs risk a allele appear to increase insulin levels (p = . ) and homa-ir index (p = . ). furthermore, this variant also seems to affect vldl levels (p = . ) and waist circumference (p = . ) in diabetic patients. the genotype analysis of rs variant demonstrated that risk g allele decreased hdl (p = . ) and increased ldl levels (p = . ), as well as affected the waist circumference (p = . ) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs a / rs g with higher homa-ir index. here we demonstrated that although both, rs and rs , adra a polymorphisms were not associated with t d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein (sip ), also known as zeb is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip mediate its binding to ebox sequences in the genome. however, there are only limited data about sip target genes. by using a home-made anti-sip monoclonal antibody, clone e , we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu , plc/prf/ and sk-hep- and found receptor tyrosine kinase-like orphan receptor (ror ) as one of the targets. sip dnabinding consensus motif cacctg was found at + kb, + kb and + kb from ror transcription start site. chip experiments validated sip binding to all consensus motifs in the ror gene region. interestingly, the strongest enrichment was at + kb suggesting that long-range interactions play an important role in the regulation of ror by sip . sip knockdown by shrna in high-sip expressing snu cells resulted in the repression of ror expression. ror is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror -positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip and ror act in similar pathophysiological processes. our finding that ror is regulated by sip at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein (sip ), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip in hcc are yet to be identified. we conducted a chip-seq study in high-sip expressing hcc cell line snu by using a home-made anti-sip antibody, clone e . among annotated genes, we selected six for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip dna-binding motif cacctg was found at - kb from transcription start site of six gene. chip qpcr experiment validated sip binding to this region with , fold enrichment. compared to healthy liver, six transcripts were upregulated in of hcc cell lines included in this study. knockdown of sip by shrna in snu cells caused upregulation of six . immunohistochemistry studies in hcc tissue arrays showed increased expression of six in tumors and inverse association with sip expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip and six in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by -hydroxy vitamin d ( (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were healthy women in age - years. the bmd was measured as a t score in the lumbar spine (l -l ) with dexa. all subjects had normal t score between (- . to . ) sd. vitd was measured by lc- -at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was ( . ae . ) lg/l, mean plasma ipth level was ( . ae . ) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = À . ). genotype distribution and allele frequency of subjects were as follows: ( %) ff), ( %) ff, ( %) ff genotype in vdrf -fok gene, ( %) bb, ( %) bb, ( %) bb in vdrb-bsmi gene. allele frequencies were f: %, f: %; b: %, b: %. when fok and bsmi were combined, %(ff-bb) and % (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > . ). but foki was not (p = ). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women ( %). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p- . . - determination of hb a c values in beta thalassemia f. g€ uzelg€ ul , g. s. seydel , a. e. yalin , e. s€ onmez , k. aksoy c ß ukurova university, adana, nigde university, nigde, mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected ml of whole blood k edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a c values were measured using the agilent hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs - /ivs - , one ivs - /ivs - , one ivs - /ivs - , two ivs - /ivs - , two ivs - /ivs - , one fsc /fsc , one fsc /fsc , two - /- , two cd /cd , one cd - /cd - , one cd - /cd - , two cd /cd -g/ cd /cd -g, two ivs - / ivs - , one ivs - / ivs - , one ivs - /fsc , one ivs - /cd , one ivs - /cd , one ivs - /cd - , one ivs - /ivs - , one ivs - / ivs - , one ivs - / ivs - , one ivs - /cd and one fsc /cd . cases were classified as diabetic ( ), prediabetic ( ) and non-diabetic ( ) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in samples from . we observed positive correlation between expression of aurc and braf (rs = . , p = . ). discussion and conclusion: previous investigation showed, that for normal prostate tissue % of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to . . based on its genome sequence, metabolic pathways for proteases, amylases, cellulases, lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of . to . makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, . and ph . . for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p- . . - biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u mg and u as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk . mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc -a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk levels in u mg were reduced by %. due to mts results, ≤ mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk level in u mg and u cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately % calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d ; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p- . . - apj receptor a c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than mm to allow rapid penetration of the fixative put in % buffered formalin for hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = ) and sinus rhythm (n = ). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu- plus platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among . transcripts representing . well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ . ) with a p-value ≤ . . results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr - and environmental pigment-producing s. marcescens strain sm . bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm . cluster has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only % of identity to xantholipin biosynthetic gene cluster. clusters and of nrps genes of s. marcescens sm did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr - contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p- . . - the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to - months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: ) active animals ) hibernated animals and ) animals immobilized for weeks in laboratory, were examined. rna libraries have been sequenced using hiseq illumina platform. coupled with genome dna sequencing provided x coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e -ubiquitin ligases murf and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. - - . in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain , and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis wild-type strain forms biofilm with its maximum on th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of . - . . the temperature optimum is in the range from °c to °c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average % lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain - , - and °c - °c respectively. the crataegus genus which is a member of rosaceae family, has approximately species worldwide and species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: , diphenyl- -picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities ( . ae . % and . ae . %, respectively), at lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values ( . , . , . ) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por gene expression, encoding vdac should increase in the stress conditions. the promoter of the por gene was used to construct some new expression systems based on yl w . a new genetic construct bearing a reporter bgalactosidase gene under control of the por promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h o and methyl viologen treated cells b-galactosidase activity increased . - . -fold reaching its maximum in the cells, grown at ph of . . thus, we demonstrated high inducibility of the por promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no - - mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine- -carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn , respectively. gene deletion was accomplished through double crossover. a cassette containing the~ bp ' and ' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn d strains isolated the achad ngn d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn d, shows azc toxicity, suggesting that ngn is a true orthologue of mpr , able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c h zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at . a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant ( . . . ). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t polymerase primer for cdna synthesis. microarray represents probes for orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps , which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane- -monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose- -phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example , -dienoyl-coa reductase, -ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of patients; healthy pregnant women (control group) and pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba c levels and homa-ir were significantly higher in gdm group compared to the control group (p = . , p = . , p = . , p = . , respectively). serum adiponectin levels were significantly lower (p < . ); whereas serum resistin levels were significantly higher (p = . ) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic , ic , amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p- . . - functional polymorphisms of ephx in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec . . . ) is encoded by ephx and catalyses the degradation of endogenous fatty acid epoxides generated by cyp epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx gene that affect the seh activity may alter the circulating levels of eets. k r and r q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k r and r q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k r polymorphism were . % wild type (aa) and . % polymorphic genotype (ag+gg) and for r q polymorphism . % wild type and . % polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a g, mtrr a g and mthfr c t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from pregnant women. the main group consisted of women (n = ) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = ) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was . ae . years compared with control of the age . ae . . the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a g (or = . , ci % = . - . ; v = . , p < , ). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr rk project number). p- . . - a study on the association between rs polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is . % above the age in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs g/c single nucleotide polymorphism (snp) is found at position À , in promoter region. the presence of a c allele for rs is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs polymorphism of the ctgf gene. study population consisted of patients with pes and controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs polymorphic allele g was . in patients, and . in controls ( . , p = . ). distribution of genotypes was gg: . %, gc: . % and cc: . % among patients, while gg: %, gc: . % and cc: . % (or = . , p = . ) in controls. statistical analysis showed that there is no significant relationship between ctgf rs snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs snp and pes. this work did not point out a role for ctgf rs in the risk for pes. a significant relationship might be found when the study population is enlarged. p- . . - evaluation of rs single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs snp of clu gene have a role in the development of pes. study population consisted of patients with pes and controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was . and that of controls was . ( . , p = . ). the distribution of genotypes was cc: . %, tc: . % and tt: . % among patients while cc: . %, tc: . % and tt: . % ( . , p = . ) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp and to evaluate the difference at mrna and protein expression levels of the tp target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp mutation. to show the tp mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of hpv negative hnscc patients. employing different databases we constructed the tp regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh was significantly associated with tp mutational state. ezh is a member of the polycomb group protein enhancer zeste homolog which is known to be directly repressed by tp and indirectly by the activation of mir- a and mir- b. we found a significant up-regulation of ezh that depend from tp mutation. it is important to understand the difference in mrna and protein expression of tp regulatory network that could depend from its mutational state. this finding suggest that ezh might be a potential therapeutic target for hnscc. p- . . - next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of all patients. for this, we used sanger sequencing of the products of multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after days of treatment. for this purpose, we performed ngs of libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to per , and , cells) for a calibration curve generation. using this approach, we analyzed all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found leukemic cell per , analyzed cells. znf is considered as a transcriptional target for p and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs , rs ) of znf were investigated in kazakh population in this study. the present case-control study was carried out with participation of kazakh females with bc and cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/À), progesterone receptor (pr+/À) and human epidermal growth factor receptor (her +/À) status were estimated. pearson p-value, odds ratio, % confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs locus in znf between the patients and control groups (p = . for allele; p = . for genotype). moreover, significant association with bc was revealed for rs after dividing patients according to er+/ À, pr+/À and her +/À status of the tumor. the g allele was associated with er+ (p = . , or = . , %ci: . - . ), pr+ (p = . , or = . , %ci: . - . ) and gg genotype with her -bc carriers (p = . , or = . , %ci: . - . ). also, g allele can be considered as a risk factors in er+/ pr+/her -luminal type of tumor (p = . , or = . , % ci: . - . ). our findings correlate with the data of several gwas where the association of the rs polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs as a marker of bc risk with predictive value, restricted to er, pr and her status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr (rs ), tox (rs ), map k (rs ), lsp (rs ) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included bc patients and healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with % confidence intervals ( % ci) were applied to data analysis. according to obtained results combinations of allele c of tox rs and a of map k rs (p = . , or = . ), also allele c of tox rs and c of lsp rs (p = . , or = . ) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox rs contribute significantly to bc risk with p-value = . , or = . . on the contrary, tt genotype of tox rs with p = . , or = . and its combination with allele t of lsp rs with p = . , or = . determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp rs and a of map k rs was found in kazakh population (p = . , or = . ). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf- ) and mouse fibroblast (l ) cell lines. cells were studied four different concentrations ( , ; ; ; lm). xtt ( , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide) protocol was applied after and hours. in our study % fetal bovine serum (fbs), % l-glutamine, iu/ml penicillin and mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in % humidity and % co incubator at °c. anti-cancer activity of synthesized complexes were determined on mcf- and l . in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control ( -fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on gem/cistreated and pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm rs , ercc rs , ercc rs , and xrcc rs . values ranged between and . ; cutoff value of . had sensitivity of . and specificity of . . patients with higher score had shorter progression-free and overall survival (p < . ). in the validation group, positive predictive value was . and negative predictive value was . . model predicting outcome of pmx/cis chemotherapy included crp level, mthfd rs , and abcc rs with scores ranging between and . . cutoff value of . had sensitivity of . and specificity of . . patients with higher score had lower probability of good response and shorter progression-free survival (p < . ). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p- . . - evaluation of anti-diabetic potential of circiliol and circilineol using caco cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt (sodium-glucose transporter- ) and glut - and glucose transport. neither ag activity nor sglt activity was increased with either circiliol or circilineol treatment in caco cells compared to positive control. similarly, neither the activity nor the expression level of glut *- was increased in caco cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak z and paubap fbe the purpose of this study was to characterize and assess the impact of a novel magnetite (fe o ) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe o nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe o @e) were characterized by ir, sem, tga and hr tem. one laboratory and p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics ( , , and hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges - nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe o @e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p- . . - specimen processing and handling for plasma ammonia measurement b. sarac ßligil , , s. abus ßo glu , f. aky€ urek , b. € ozt€ urk selc ßuk medical school, konya, biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at °c) within minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at °c) within minutes and analyse refrigerated - °c hours (a , b ) and hours (a , b ). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results ( . versus . lg/dl, p < . ). in all protocols including a , a , b , b also cause an elevation in plasma ammonia results ( . , . , . and . ; p = . , p < . , p < . , p < . , respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb (gene encoding b-catenin; mostly mutations in exon ) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with ll of extraction buffer at °c over night. the tubes were boiled for min to inactivate the proteinase k. ctnnb exon was amplified by pcr. sscp is used to observe any difference between the groups. genomic dna was isolated as described above. ll of each pcr products mixed with ll denaturating buffer and were denatured by heating at °c for minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on % acrylamide/ bis gel in . tbe buffer for . hours at v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between and % in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb exon between the tumor group and normal groups. p- . . - theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol , b. uc ßar koc ß university, istanbul, american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe , tyr -octreotate, tate) has been labelled with the positron emitter ga and the beta and gama emitter lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ ga] dota -tyr -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to mm. from mm. in d.-after fifth operation). due to its high sstr selectivity, [ lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p- . . - cardiovascular diseases can be treated by using 'tetr-odd-vp ' and 'hre' hypoxia inducible systems a. celik , t. kaya , s. cigdem , m. g€ und€ uz department of medical genetic, turgut ozal university, ankara, faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif- a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif- a protein levels increase under hypoxic conditions as a result of decreased o -dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif a between and in front of tetr dna binding domain and vp transactivation domain, so that tetr-odd-vp or odd-tetr-vp could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp and odd-tetr-vp constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p- . . - osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a d cell/ scaffold bioconstruct to a continuous magnetic field during weeks of osteogenic induction. in this view, mc t -e murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc t -e cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc t -e cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p- . . - impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = , . ae . yr, . ae . kg) and untrained (n = , . ae . yr, . ae . kg) subjects performed three soccer matches ( min) in field ( m versus m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < . was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < . ). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < . ) in trained ( . ae . u/l versus . ae . u/l, % (m) . ae . u/l versus ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n) and also untrained groups ( . ae . u/l versus . ae . u/l, % (m), . ae . u/l versus . ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p- . . - regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in subjects ( males and females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between and , were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef . %, gk . %, m . %, r . %, s-l . %, s . %, ehsani . % and sirdah . %. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, out of , out of and out of patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i- (g>a), ivsii- (g>a) and heterozygous codon deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p- . . - novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev , l. gera , r. hodges institute of molecular biology and genetics, kyiv, ukraine, university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm- appeared to be the most effective, with ic values of lm and . lm in rat glioma c and human glioblastoma u cell lines, respectively. bkm- suppressed erk / and akt phosphorylation in u cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm- and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi l inhibited the viability of u cells more effectively than tmz. furthermore, the combination of chi l and bkm- resulted in an additive cytotoxic effect. chi l -mediated decrease of cell viability was associated with a g /s transition arrest. chi l provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d expression, as well as a substantial increase in p level. in addition to the accumulation of p , we observed the upregulation of cdk inhibitor p . therefore, g /s arrest in chi l -treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p . harmful hereditary mutations in brca and brca are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca and brca genes in breast cancer-diagnosed patients, that one of them is male, and person yet healthy whose brca was sequenced by sanger sequencing. the data were analyzed by using seqscape software . and detected variations were compared with literature. in brca , we determined different benign genetic variations and variation with unknown significance and variation which has not in literature. in brca gene of patient and healthy person, benign variations, variations with unknown significance, variations which has not in literature and mutation were determined. this mutation is c. - delgtca and is located in occr. c. - a>c variation in brca gene, was determined in only male patient.c. t>a variation in exon of brca , was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p- . . - inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi x . non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi x . introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included adult male rats, divided into groups. each group included rats. i.group: control (regular feed), ii.group: cpe ( . mg/kg/day), iii.group: rw ( mg/kg/day) and iv.group: cpe ( . mg/kg/day) + rw ( mg/kg/day). following days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn + pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm À ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h dcf-da. gsh level was estimated using lowry method. an intracellular labile zn + pool was assessed using fluozin- -am. investigations were performed on the specord m , hplc system lc- prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn + in erythrocytes of patients with ah + dm + compare with ah + dm À and ah À dm À was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn + level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm À , ah À dm À ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, , : - ; lipid insights, , - ] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., , : - ] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde - -hexadecanal. it was found, that -hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal (suppl. ), abstracts: mem. biol. s , lipid signaling & dynamics, p. .] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the - - proteindependent inhibition of apoptosis signalregulating kinase protein kinase ask (apoptosis signal-regulating kinase ) is a member of the mitogen-activated protein kinase kinase kinase (map k) family that plays a crucial role in immune and stress responses. the activity of ask is regulated through homo-oligomerization and interaction with other proteins including the - - protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask is dephosphorylated at ser and the - - protein dissociates. this dissociation is then one of the factors that lead to the activation of ask . we performed low-resolution structural analysis of the kinase domain of ask (ask -cd) bound to - - using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask -cd binds to the - - protein in two to two stoichiometry through a small binding interface involving surface of - - outside its central channel and several regions from the c-lobe of ask -cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask -cd to - - modulates conformation of ask 's activation segment. these results suggest that the - - binding suppresses the catalytic activity of ask through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask and - - and offers a plausible structural explanation for the - - protein-dependent inhibition of ask kinase activity. introduction: thymoquinone ( -methyl- -isopropyl- , -benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c rat glioma cells functioning was studied. results: it has been shown that the cultivation of c cells with tq in concentrations of - lm during hours strongly inhibits cell proliferation and induces cell death with id of lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c cell death by %. we analyzed mapk pathways involvement in c cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd and jnk pathway by sp does not influence on tq-induced effects. on the contrary, the specific phosphoinositide- -kinase (pi k) inhibitor (ly ) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = ) and patients with co-infection (hiv and hepatitis b and / or c) (n = ). as a control we used the data of healthy women (n = ). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control ( . times; p = . ) and group with hivmonoinfection ( . -fold; p = . ). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control ( . times (p = . ) and . -fold (p < . ), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > , ). total antioxidant activity parameter decreased . fold (p = . ) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = . , compared to the control and p = . , compared with the group with hiv-monoinfection) and the level of a-tocopherol ( . -fold to control and . fold to hiv-monoinfection) was detected in the group with hiv-coinfection. . -fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p- . . - acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose groups were formed. rats in group served as control group animals which were only given corn oil. group , group and group were administered , , mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group , group and group were inhibition percentage of %, %, and % respectively, comparison of group 's che activity. the liver alt, ast and ldh increased in group and group compare to group and group (p < . ). we also observed that there was vacuolar and hydropic degeneration in liver of group . according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised patients with ami and healthy subjects. patients that had an acute myocardial infarction (ami) less than hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon dhc were significantly higher in group with ami (p < . , respectively p < . ). elevated levels of mda (p < . ) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < . ) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p- . . - dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet , which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft , the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet gene. however, we found that a strain lacking fet is more sensitive to cu surplus conditions than a strain carrying the aft gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet gene expression. we next evaluated whether ace could be the alternative regulator of fet under cu excess. to test this hypothesis we first constructed the double mutant aft ace and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft or ace mutants. we next monitored the expression of fet gene in cells lacking ace , using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet expression is dependent on ace when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet by ace in an aft independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon- derived from human long bones (atcc Ò crl- tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with %, the superoxide anion is reduced with % compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = ) and age matched control group (n = ) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h o -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss software package. results: dna strand breaks and h o -induced dna damage were found to be higher (p = . for all), the ratio of gsh/gssg was found to be lower (p = . ) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h o -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = . ). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = . ). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p- . . - validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product iso-prostaglandin f a in urine m. kant , m. akıs ß , h. _ is ßlekel , department of medical biochemistry, school of medicine, dokuz eylul university, izmir, department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey -iso-prostaglandin f a ( -iso-pgf a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of -iso-pgf a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version . were used for data analyses. mrm transitions used were m/z? / for -iso-pgf a and m/z? / for -iso-pgf a-d . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for -iso-pgf a was determined as ng/ml by using standards ranging from . - ng/ml. analytical sensitivity of the method was determined by lod with s/n of and loq with s/n of . lod and loq for iso-pgf a were . À and À ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = ). the intra-and interday cvs for -iso-pgf a were . % and . %, respectively. sid-mrm lc-ms/ms method for absolute quantification of -iso-pgf a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p- . . - synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase / activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g /g peak, an increase in the annexin v(+) cells and the cleavage of caspase- and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl- ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto- h cells, cis/res was less efficient in killing h- cells. h- cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin- and lc a. inhibition of autophagy by bafilomycin a rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h- cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. adult male mice ( - weeks) were randomly divided into groups: control group received normal saline ( . ml/day, ip), cp group received cp ( mg/kg/week,¬ ip), the cp+ep group received ep ( mg/kg/day, ip) along with cp, were treated for days. mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < . ). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < . ). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < . ). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < . ). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < . ). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of mg/kg (group ), mg/kg (group ) and mg/kg (group ). only plain corn oil was given to control group (group ). the rats were sacrificed hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group compare to group , besides the insulin levels of group and group were significantly higher than group (p < . ). pancreas amylase and lipase activities significantly decreased in group and group compare to group and group (p < . ). however, there was no significant change in pancreas mda and tos levels (p > . ). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < . ). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < . ). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb ( mg l- and . mg l- ) for hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. day). ml physiological saline (% . nacl solutions), mg/kg/day paracetamol, mg/kg/day paracetamol and mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p- . . - lipid rafts are involved in modulation of ca + responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim Ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan Ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca + concentration ([ca + ] i ) increase due to ca + mobilization from thapsigargin-sensitive ca + stores and subsequent store-dependent ca + entry in rat peritoneal macrophages. it is known that key proteins involved in ca + signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca + ] i in macrophages. [ca + ] i measurements were performed with fura- am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca + ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with mm mbcd for hours before molixan addition causes significant inhibition of both ca + mobilization (on average, by . ae . %) and subsequent ca + entry (on average, by . ae . %), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca + responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca + ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range ( to lm) against phenotypically different mcf- , mda-mb- and sk-br- breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p , p , p and/or p . apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of lm, whereas at the concentration of lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut- and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h ax phosphorylation and bp foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, / /d/nz / . nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn that is accompanied by the relocation of nucleolar proteins and p -based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf- , mda-mb- and sk-br- ). we found that low dose nutraceutical treatment resulted in p -mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt , dnmt a and dnmt b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, / /d/nz / . p- . . - rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < . ), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < . ). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < . ), while malondialdehyde (mda) levels increased notably (p < . ). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type diabetes mellitus (t dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t dm imply an increased enzymatic activity of pa ( . %), which correlates with increased levels of ea ( . %) and glycated hemoglobin (hba c) ( . %), compared with control group. the variables hba c, pa and ea are correlated: the identified pearson correlation coefficients r = . and r = . respectively, have an associated probability of p < . and p < . a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi , e. demir , k. cinar gaziantep university, school of medicine, gaziantep, haran university, sanliurfa, department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group (ir) received gy of gamma irradiation as a single dose to total cranium plus ml saline daily. group received daily plain dmso. group received daily plain saline. at the end of the day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p- . . - role of leptin and adiponectin in obesityassociated oxidative stress e. becer , a. c ß irakoglu near east university, nicosia, cyprus, istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included obese and non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < . ) and adiponectin (p = . ) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < . ) and malondialdehyde (p < . ). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = . ) and malondialdehyde (p = . ) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around %. an extract from aronia showed greater than % efficiency, and a net ethanol extract from all the berries showed a % effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega / / and / / . p- . . - attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins -e / pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins- e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene ( lm, hours) application have been made after resveratrol ( lm, hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins- , ins- and sirt- were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo- gene was increased while pdx- was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, adult sprague-dawley male rats were divided into groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for minutes followed by a hour of reperfusion. in the treatment group, days before ischemia mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as % in the control group, whereas it was % in the i/r group. this value was decreased to % in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost % efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak ( %) at a concentration of mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to %. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega / / and / / . the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t d wt and gh+ cells. although forced gh expression induced cell proliferation, lm curcumin inhibited cell invasion. curcumin ( lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t d cancer cells. although curcumin decreased az expression in t d wt cancer cell, increased az expression was determined in t d gh cancer cell. pao and ssat expressions were upregulated in t d gh+ cells. concomitantly, putrescine levels were increased in t d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak research project (project no: z ). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. - weeks old female sprague-dawley rats (n = ) included in current study were divided into groups: control group (n = ) with healthy rats; breast cancer group (n = ); breast cancer group (n = ) treated with mg/kg paclitaxel injection (once a week for weeks); breast cancer group (n = ) treated with mg/kg royal jelly (by oral gavage for days); and finally breast cancer group (n = ) treated mg/kg royal jelly in addition to mg/kg paclitaxel injection. rats with breast carcinoma was obtained at th days after a single dose injection of mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < . ). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > . ). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase (pon ) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese ( g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = ). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups ( rats in each group). group : sham + saline; group : anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group : anp + gallic acid ( mg/kg/day, intraperitoneal). at the end of th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin- (il- ), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il- , plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < . ). however; tas and nitrite + nitrate levels did not show any significant difference (p > . ). on the other hand, while serum amylase, il- and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > . ). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p- . . - evaluation of oxidant/antioxidant system parameters, il- and il- levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder , s. bahsi , t. bahsi , v. topc ßu , a. bakir ankara universty faculty of medicinel, ankara, zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il and il levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from pregnancies affected by down sydrome and normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il and il levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < . ). besides this, in study group, cat and il- levels are found singnificantly lower than control group (p < . ). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il- levels (p > . ). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g cells. cytotoxicity of linalool was assayed by celltiter-blue Ò cell viability assay. malondialdehyde levels result in membrane damage in hep g cells were assayed by using fluorometric method. hep g cells were incubated with linalool at , and hours. the viability of hep g cells decreased in a manner dependent upon concentration and incubation time. the ic values were calculated . lg/ml ( hours), . lg/ml ( hours) and . lg/ml ( hours). but, cell viability of hep g cells increased when the cells preincubated with linalool at lower concentrations (˂ic ) against h o cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h o was decreased when the cells preincubated with linalool before h o incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included subjects ( female and male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and nd, th and th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = . and . , respectively). thiol levels decreased significantly in both gender at postprandial nd, th and th hours as compared with fasting condition (p = . ). while tbars levels increased at postprandial nd hour, that was only significant for male individuals (p = . ). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p- . . - ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in patients with prediabetes and controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p- . . - the effects of s _ io nanoparticles of rat uterine smooth muscle specially used in textile field sio nanoparticles on uterus smooth muscle was aimed to be researched. in this study wistar albino female rats were used. rats were separated in groups as control, dose ( lg/ml), dose ( lg/ml) and dose ( lg/ml). nanoparticle's size was chosen as nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio nanoparticles, in nm size and in , and lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx )] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine -epi-prostaglandin f a ( -epi-pgf a)] were determined. the results were compared with those of controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher ( % for cuzn-sod, p = . ; % for cat, p . ) and the levels of lipid peroxidation biomarkers were lower (% for mda, p = . and % for -epi-pgf a, p > . ) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = - . , p = . ). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p- . . - investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, patients with acute limb ischemia (li group; mean age years) and healthy individuals (control group; mean age years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < . ). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase activity (pon ) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on women consisted of patients with pcos and healthy ones as control. serum pon activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at nm. molar absorptivity coefficients were used in the calculation of pon and are activities as nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group ( . ae . - . ae . u/ml p < . , . ae . - . ae . u/ml p < . , recpectively). the pon activities are higher in the patient group ( . ae . u/ml) compared to the control group ( . ae . u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were . ae . absu, ima levels of control subjects were . ae . absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < . ). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < . ). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p- . . - the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard ( % protein, % ( % sucrose) carbohydrate, % lipid) and sucrose ( % protein, % ( % sucrose) carbohydrate, % lipid) diet fed bdnf heterozygous mice liver tissues. the male c bl strain wild type (+/+) and bdnf heterozygous (+/À) mice ( weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for weeks. first group involved in c bl wild type mice and fed by standard diet. second group contained c bl bdnf heterozygous mice and fed by standard diet. third group consisted c bl wild type mice and fed by sucrose diet. fourth group involved in c bl heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c bl/ mice inbred strain wild type and bdnf heterozygous (+/À) mice. animals were divided to two groups: wild type (n = ) and bdnf heterozygote mice (n = ). the animals were fed ad libitum by high-fat diet during month. weight gain was recorded every th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p- . . - disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p- . . - determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p- . . - effects of alpha- antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha- receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha- antagonists on oxidative system of rat heart tissue. male wistar albino rats were divided into groups randomly. ) tamsulosin ( mg/kd/day), ) terazosin ( mg/kg/day), ) doksazosin ( mg/kg/day), ) alfuzosin ( mg/kg/day), ) control. all drugs were administered every other day single doze via oral. rats were sacrificed after days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = . ). in addition, tamsulosin group and terazosin group were also significantly different (p = . ). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. wistar albino rats were divided into groups randomly. control (i.p. physiological saline), sbt (i.p. mg/kg/ hours sbt), stress (i.p. physiological saline; hours cold + immobilization stress) and sbt + stress (i.p. mg/kg/ hours sbt; hours cold + immobilization stress) groups were formed. nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = . ), total glutathione (p = . ) levels and glutathione peroxidase activities (p = . ) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = . ) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker -nitrotyrosine level was found to be significantly higher when compared with control group (p = . ) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. children and adolescents with obesity (age: - ); and age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics ). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity ( . ae . lmol/l; . ae . nmol/ml) than the controls ( . ae . lmol/l; . ae . nmol/ml) (< . ). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p- . . - relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats ( - g) received three i.p. injections of taa ( mg/kg) at hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca + chelation had almost the same effect as pharmacological nos inhibition suggesting that ca + -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh ) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries ( , and mg/kg) by oral gavage during days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < . ). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < . ). the maximum hypoglycemic and antioxidant effects were observed at mg/kg dose of mc. background: human serum paraoxonase (pon ) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon prevents ldl oxidation by hydrolyzing lipid peroxides. pon is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon q r isoenzymes. methods: pon q r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with mm edta for minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of mm edta to partially purified hdl-pon q r isoenzymes inhibited % of htlase and arylesterase activities. inactivation of pon for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca + -dependent inhibition of pon q r arylesterase/htlase by using the metal chelator edta, did not alter pon 's ability to inhibit ldl oxidation. pon 's ability to protect ldl from oxidation may not require calcium. p- . . - evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad , college of applied medical sciences, aljouf university, aljouf, saudi arabia, faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type diabetic patients. a total of type diabetic patients and healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx- ). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type diabetic patients than the control group (p < . ). one of the boron compounds is sodium perborate tetrahydrate (nabo . h o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose- -phosphate dehydrogenase (g pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < . ) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan , e. aky€ uz , g. baysal furtana , s. s. ellialtioglu , r. tipirdamaz nezahat g€ okyigit botanic garden, istanbul, department of biology, faculty of sciences, gazi university, ankara, department of horticulture, faculty of agriculture, ankara university, ankara, department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h o ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including mg/l , -d and . mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing and lm h o for hours and then transplanted on the mediums including mm nacl for hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h o -only and h o pre-treated tissues. pre-treatment with h o decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h o pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. the result showed pre-treatment of lm h o induced acclimation of the plants to salinity. in addition, lm h o pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type (cb ) receptor agonist acea, the antagonist am- and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study male wistar albino rats were used weighing - g. each group was consisted of rats. study groups: : control, :penicilin( iu), :penicillin( iu) + ghrelin( lg), :penicillin( iu) + am- ( . lg), :penicilin ( iu) + acea( . lg), :penicillin( iu) + am- ( . lg) + ghrelin ( lg), :penicillin( iu) + acea( . lg) + ghrelin( lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule- (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of patients with a diagnosis of as according to newyork ctriteria and control subjects were included in our study. serum endocan, interleukin- (il- ), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il- , tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < . ). whereas, serum il- , tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il- , tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = . , p = . ; r = . , p = . ). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p- . . - investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged or hours according to parasitemia state ( - %). biochemical analyses were performed in erythrocyte culture in which parasitemia between % and %. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed times with physiological saline, erythrocyte suspensions were stored at- oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < . ). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g pdh, gshpx, gshrx p- . . - in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut , g. kanbak istanbul bilim university vocational school of health services, istanbul, eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol ( mm, mm ve mm) alone and together with lg/ml aspirin, and possible protective role of mm betaine on rat brain synaptosomes. male sprague dawley rat forebrains were divided into equal pieces and pooled to form study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < . ) was observed in mda level of mm ethanol group compared to control group. different doses of ethanol ( mm, mm ve mm) + aspirin exposure significantly increased (p < . ) mda levels compared to controls, whereas betaine administration significantly decreased (p < . ) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p- . . - analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif- a in hepatocellular carcinoma cell line, hep b under hypoxia. method: hep b cells were subjected to normoxia ( % o ) and hypoxia ( % o ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif- a, pgc- a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at lm. bbr treatment has shown significant reduction in hif- a and sirt which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt expression. bbr induces the overexpression of sirt and its deacetylated-pgc- a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif- a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with lm h o after minutes lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h o . also our data suggest that ziziphus jujuba is effective in preventing h o induced oxidative stress and nitric oxide levels. discussion: some research showed that h o was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h o . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the -hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at + . v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p- . . - impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller , h. budak , m. sisecioglu , m. c ß iftci department of chemistry, faculty of science, atat€ urk university, erzurum, department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose- -phosphate dehydrogenase (g pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: / ). p- . . - protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur ( , and mg/kg) were administered orally for days. on the sixth day, chlorpyrifos ( mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < . - . ). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < . ). cur administration reduced all of these changes (p < . - . ). similarly, cur at the doses of mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < . ). additionally, the histopathological damage scores induced by chlorpyrifos (p < . - . ) were decreased by the administration of cur (p < . - . ). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after g ogtt which was applied in pregnant women for screening. methods: the serum specimens of pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = ) and prediabetic pregnant women (n = ) in the th and th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the th and th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between th and th minutes regarding the xo enzyme activity in normal pregnant women (p = . ). in normal pregnant women, the median xo enzyme activity in the th minute was . ( . - . ) u/l and it was . ( . - . ) u/l in the th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in th and th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased . times under red and blue lights and . times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was . times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p- . . - effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c glioma and mouse n a neuroblastoma cells and incubated for hours. at sub-lethal concentrations ( , , lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein (gap ) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c cells, and of nefh and biii tubulin in n a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at g for minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h o absence and presence of curcumin ( - lg/ ml) for hours at °c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase , and activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h o ) on hepg liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg cells's - - hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg cells were determined with flow cytometry method with the help of annexin v-apc and aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: hours ic > lg/ml, hours ic . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; hours ic = . lg/ml, hours ic = . lg/ml, hours ic = . lg/ml. increasing concentrations of ehta extracts were determined to direct hepg cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of mg in g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg was cytostatic at low doses, and cytotoxic at high doses. p- . . - relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of patients ( subject as scf and subjects as ncf) were analyzed. ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group ( ae versus ae ng/ml, p = . ). also there was a significant correlation between serum cp levels and tfcs (r = À . , p = . ). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t ) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n ), and experimental (n ) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t ( mg/ g) in postoperative day. rats were sacrificed on postoperative day . serum thyroid stimulating hormone (tsh), free t (ft ), and free thyroxine (ft ) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: . ) and centrifuged at rpm for minutes ( °c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t hormone had not decline in ft levels compared with the control groups. a significant decrease in ft levels was found in t given rats on postoperative day . whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft level. although tos levels and osi values were similar in both groups, t supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip (tat interacting protein, kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to . hour of lschemia and hour of reperfusion, group iii of rats that received intraperitonealy curcumin ( mg/kg) . hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase , and were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase- and ieveis, and increased the uterine tissue levels of tac but, caspase activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group ( . ae . %) and decreased after treatment with curcumin ( . ae . %). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p - on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, -day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were , , and days old. their weight and tumor diameters were measured throughout the months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p- . . - effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e ) in food preservation for approximately years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf- , colon cancer cell line, ht- , lung adenocarcinoma cell line, a , and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with % heatinactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ ml, ug/ml, ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over hours. according to our results, ha induced a decrease in ht- , a and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf- cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf- cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p- . . - chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using male wistar albino rats weighing - g. rats were divided into five groups (n = / group). group i comprised the control group, group ii was exposed with saline + immobilization stress ( minutes daily immobilization stress for days and . ml saline was administered perorally minutes before immobilization), group iii was exposed amisulpride ( mg/kg/day) + immobilization stress, group iv was exposed fluoxetine ( mg/kg/day) + immobilization stress and v. group was exposed amisulpride ( mg/kg/ day) + fluoxetine ( mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < . ). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < . ). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < . ). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > . ). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p- . . - protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against -methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin , g. oto, f. g€ ok, y. karakus, d. yildiz y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st -methylcholanthrene ( -mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % . nacl, group ii: the b(a)p were administered mg/kg via ip. four times. group iii: the -mc-treated animals were administered mg/kg via ip. four times, group iv: ba was given mg/l/day with water. group v: bx was given mg/l/day with water. group vi: b(a)p mg/kg via ip four times + ba mg/l/day dosage with water. group vii: -mc mg/kg via ip four times + ba mg/l/day with water. group viii: b(a)p mg/kg via ip four times + bx mg/l/ day dosage with water. group ix: -mc mg/kg via ip four times + bx mg/l/day with water. the experimental period was continued for days. statistical analysis showed that the -mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < . , p < . ,p p- . . - effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed - g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to mg/kg body weight alcl in drinking water for and weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., tacal, Ö., take, g., takic, m.m., taldykbayev, z., talim, b., tamashevski, a., tamer, f., tamer, l., , , taneva, s., taniyan, g., , tanrisev, m., tanriverdi, e.c., tanriverdi, k., tarhan, m., tarhan, t., tartar, s., tas, a., , taskin, a., taskin, t., taskiran, e., taskiran, b., taskoparan, b., taspinar, r., , tastan, Ö., tatli, Ö., tauraite, d., tay, t., tayman, c., taysi, s., , teker, h.t., tekes, s., tekin neijman, s., tekin, m.h., , tekin, g., , tekin, m., tekin, n., tekin, g., telci, d., , telefoncu, a., temel, h.e., , , temelie, m., temizgül, r., temlyakova, e., teplova, v., terashima, r., tercan avci, s., terekhov, s., tereshenko, o., terzi gulel, g., terzi, e., , terzi, e., terzioglu, g., terzioglu, o., testoni, g., tetik vardarli, a., tevdoradze, t., tevzadze, l., tezcan, Ö., tezcanli kaymaz, b., thielens, n., thomaidou, d., thomas, a., thornton, j.d., ticea, a.c., tikhonova, a., tileva, m., , timofeev, v., , timofeev, v., timofeeva, e., tipirdamaz, r., tiryaki, m., , tok, m., tokay, e., toker, a., , tokgun, o., toksoy Öner, e., tokyol, Ç., toman, r., tomasi, a., tombul, n., , tooke, c., topaloglu, h., topbas, m., topcu, b., topcu, c., topcu, g., , , topçu, v., topcu, c., topçuoglu, c., , topel, h., , toprak, b., toprak, m.s., torac, e., tosner, z., tosun, m., , toy, h., toymentseva, a., tozkoparan, b., trabulus, d.c., trantirek, l., trantirkova, s., trchounian, a., , trizna, e., , troshagina, d., tro t, m., truncaite, l., , tsakalidou, e., tsarkov, d., tsarkova, m., , tsigara, e.g., tsigara, m.g., tsverava, l., tsvetkova, e., tsyba, l., tsyganov, d., tsymbal, d., tüfekçi, a.r., , tufekci, a.r., tufenkci, h., tuglu, m.i., , tuglu, i., tuli, a., , , , tuli, a., tulubas, f., tunali, g., tunca gedik, s., , tunca gedik, s., tunçdemir, m., tuncel, h., tuncel, h., tunçer, s., tuncer, e., , tuncer, z.s., tuncer, b., tuncer, e., tuncez akyurek, f., tunçez akyürek, f., tuner, h., tüney kizilkaya, i., tural, b., tural, s., turan, i., turan, m., , turan, v., turan, y., turan, c., turano, p., , türel, s., , turhan, t., , turhan, y., , turk, s., turkan, a., türkcan kayhan, c., turkekul, k., türkel, s., turkeri, o.n., turkeri, n., turkkan, b., turkmen, s., türkmen, n., turkon, h., tutar, y., tüten, a., tutkun, e., , tüylü küçükkilinç, t., tuz, m.u., twardovska, m., tyapkina, o., tzartos, s., photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf- bucharest, national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, microelements modifying oxidative stress status were determined by icp-ms/ms in the hno diluted serum samples collected from chl-positive adult females (n = ) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging - g) were randomly divided into groups, as each composed of rats. after hippophae rhamnoides l. leaf ether extracts of mg/kg, mg/kg and mg/kg doses and mg/kg doses of famotidin orally administered, mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental ( mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; hours torsion/ hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape ( lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi k, akt and mtorc. intensities were evaluated as mild ( ), moderate ( ) or strong ( ). serum ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c ( . ae . lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p- . . - binding of fas and cu(ii) ions to hsa changes its cys thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion ( . mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys -sh decreased for % after cu(ii) ion binding, and during hours incubation at °c, it was further decreased for % (stearic acid, mixfas) and % (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg ( mol/ mol hsa), lead to decrease in cys -sh content depending on fa present: %- % for myristic and stearic acid, % for oleic acid and mixfas and % for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases pirto ek p- . . - anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf- and mda-mb- background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl- is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl- levels. methods: our study group includes subjects with vitamin d deficiency (group ) and age and sex-matched healthy subjects with normal serum levels of vitamin d (group ). plasma (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl- analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > . ), plasma ykl- levels were significantly higher in group than group (p < . ). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl- levels in group . vitamin d deficiency may be related to increased ykl- levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl- , inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on unrelated cad patients and healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as . %, . % and . %, respectively. in the control group, frequencies of genotypes were found as . % for aa, . % for ac and . % for cc. we did not observe difference in apj receptor a c polymorphism between cad patients and healthy controls (v = . ; df = ; p = . ). the a allele was encountered in % ( ) of the cad and . % ( ) of the controls. the c allele was seen in % ( ) of the cad and . % ( ) of the controls. allele frequencies were not significantly different between groups (v = . ; df = ; p = . ). the frequencies of apj receptor a c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ . . in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p- . . - comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups ( rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at hours intervals, intraperitoneally (i.p.). cerulein group received cerulein ( lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide ( %), two times, at hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin ( mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < . , p < . , respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < . ). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < . ). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < . , p < . , respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < . ).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p- . . - identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx- and rtx- ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of mg/ml was prepared, cleared by membrane filter of . lm and freezed at À °c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. mg of dry extract was reacted with ll pyridin + ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including % trimethylchlorosilane (tmcs) and incubated for minutes at °c. all analyses were performed with shimadzu gcms-qp ultra by using a flame ionisation detector (fid). rtx- and rtx- ms capillary columns and helium for carrier gas at a flow time of ml/minute were used. injection was applied on split mode at °c. derivatized propolis sample was injected as ll, initial column temperature was adjusted as °c, then increased to °c with increments of °c. total analysis time was determined as minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx- column were much more than those of rtx- ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx- column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after and exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to . lg/ml of cd (cdcl .h o) and zn (zncl ), and their mixture, for , and days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph . ). homogenates were centrifuged at , g ( min, + °c). supernatants were stored at À °c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < . ). results: gst and gsh changes were recorded as decreases after all metal exposures at day . although day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor- (lox- ) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox- expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor- (lox- ) in animal model of type diabetes mellitus. methods: we compared groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz ( mg/kg). animals were kept in standard condition. in day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox- levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox- levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox- levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type diabetes. p- . . - investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur , i. ince , i. abidin , a. alver objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain animals. the rats in ns treated groups were given ns ( . ml/kg) once a day orally for weeks starting days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b ) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of , , and mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with mg/kg body wt. doses ( %). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: patients with prostate cancer and healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were . ae . lmol/l in the control group, . ae . lmol/l in the patients before rp, and . ae . lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < . ). native thiol, total thiol and tas levels were higher months after rp compared to before rp in patients, but these changes were not significant statictically (p values . , . and . respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p- . . - is there any relation between g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to pregnant women aged - years and at - weeks of gestation. plasma glucose concentrations were measured initial, hour and in addition to test hours after ingestion of g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and hour serum total antioxidant status (tas) levels (r = . ) and the oxidative stress index (osi) (r = . ) was found. there was a positive weak correlation with initial and hours total oxidant status (tos) levels (r = . ) but statistically significance difference was not found (p > . ).in this study after ingestion of g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their , -diphenyl- -picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid ( . - . -lg/g), p-coumaric ( . - . -lg/g), and total phenolic content (ranged . - . -lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at - c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after hours following cpb were significantly higher than the before surgery (p < . , p < . , p serum ngal levels in after hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = . , p < . ; r = . , p < . ; r = . , p < . ; r = . , p < . respectively). ngal levels were positively correlated with total perfusion time after cbp (r = . , p < . ).the results of our study show that, increased ngal levels hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of wistrar male rats were used. groups are named as healthy, ind control, famotidine ( mg/kg) and three different doses of tq ( . , and mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. min later, mg/kg ind administrated to each rat. animals were sacrified about hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq ( . , and mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and , diphenyl- -picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + mg/kg zn sulfate (low dose; group ), al diet + mg/kg zn sulfate (middle dose; group ) and al diet + mg/kg zn sulfate (high dose; group ). zn sulfate solutions were administered . ml/day orally for days and in day rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group and ; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group . in kidney; aopp level was decreased only in group and sod level was only decreased in group as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group ). gst level was decreased in only group conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group was composed of patients with type dm and cataract and group was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of patients, totally patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < . ). tos serum levels in dm group was statistically higher than the control group (p < . ). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > . ). group , divided into two subgroups according to their hba c levels, there was no statistically significant difference between the subgroups when hba c levels were compared with the relationship between serum and ha's tac and tos levels (p > . ). there was not an association between the gender, age and the levels of tac-tos in both groups (p > . ). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik , g. erbil , z. c ß avdar , c. ural department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to hours of ischemia followed by hours of reperfusion. ferulic acid was administered ( mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase- , inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase- and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < . ). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < . ). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < . ). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p- . . - across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [ ] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane- , p-phenol- , -p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. %) charged titanium phthalocyanine ( . , . , . , and lm) were determined on a human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc( ), tritc-phalloidin and dapi staining protocols. waltmann pdt l was used as the non-toxic light source at j/cm fluence and mw/ cm fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: rvo patients and controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > . for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of osa patients were included in the study ( controls, with mild, with moderate, and with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi< served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was . ae . years and . % ( / ) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > . ). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < . ). there was a negative correlation between ahi and serum total thiol levels (r = À . , p < . ) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera -ohdg, mda and il- were significantly higher in diabetic group than control group (p < . ). although there was a notable positive correlation between mda and -ohdg, there was no a relationship between -ohdg or mda with il- . discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il- , induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose- -phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g- -p-d) enzyme activity and the effects of quercetin on erythrocyte g- -p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = ) received physiological saline. the quercetin group (n = ) received quercetin ( mg/kg/ day) via i.g. route the alcohol group (n = ) received ethanol ( % v/v, ml/day) via i.g. route. the alcohol + quercetin group (n = ) received ml of ethanol ( % v/v) hours after quercetin treatment ( mg/ kg/day). experimental procedures were peformed for days. erythrocyte g- -p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < . ). in the alcohol group, the erythrocyte g- -p-d activity was found to be significantly decreased than those in the control group (p < . ). statistically significant differences were observed in erythrocyte g- -p-d activity between the alcohol group and the alcohol + quercetin group (p < . ).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g pd, oxidative stress, quercetin p- . . - effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = ), wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of minutes. four hours later, except control, sham-a, sham-c groups, mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca , ca , and ca region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il- b, il- , malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il- b, il- , mpo, no, and tnfa levels. it could increased il- b levels in chronic stage but not affect to il- , mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il- , and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: ( ) nitrite formation from nitrate by nar, and ( ) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-( -naphtyl)-ethylenediamine, which form red color measured at nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment ( °c, min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p- . . - the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation co) on the radiotherapeutic equipment teragam in a dose of gy and received phytomedicine eminium regelii in a dose of . mg/kg orally within days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in times in spleen, in times in thymus, in times in adrenal glands, in liver in times, in lymph nodes of small intestine it in times. mda decreased in liver up to and times, in spleen in . times, in thymus in times, in liver in times, in adrenal glands in time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p- . . - evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal , c. topc ßuoglu , m. cingi clinic of biochemistry, ankara polatli duatepe public hospital, ankara, clinic of biochemistry, ankara numune training and research hospital, ankara, clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm- ) criteria for bipolar disorder i. healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p- . . - xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in fmf patients and healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc- , sjsa as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ml, ug/ ml, ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc- and sjsa cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc- cells with an ic value of . lg/ml. contrary to this, ha induced proliferation of sjsa cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa cells with an ic value of > lg/ml. on the other hand, cell migration was reduced in pc- cell line and interestingly, migration was accelerated in sjsa cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal (suppl. ) ( )